Anti-NOTCH3 antibodies

ABSTRACT

Monoclonal antibodies that bind and inhibit activation of human Notch3 are disclosed. The antibodies can be used to treat cell proliferative diseases and disorders, including certain forms of cancer, associated with activation of Notch3.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/653,684, filed Jun. 18, 2015, which is a national stage applicationfiled under 35 U.S.C. § 371 of International Patent Application No.PCT/US2013/076615, filed Dec. 19, 2013, which claims the benefit of andpriority to U.S. Provisional Patent Application No. 61/866,787, filedAug. 16, 2013, and U.S. Provisional Patent Application No. 61/739,435,filed Dec. 19, 2012, the entire disclosures of each application beingincorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The field of the invention is molecular biology, immunology andoncology. More particularly, the field is antibodies that bind humanNotch3.

BACKGROUND

Notch pathway signaling is involved in numerous cellular processes,including cell fate determination, differentiation, proliferation,apoptosis, migration and angiogenesis. In mammals, there are four Notchproteins (sometimes called “Notch receptors”), designated Notch1-Notch4.All four Notch proteins have a similar domain structure, which includesan extracellular domain, a negative regulatory region (NRR), asingle-pass transmembrane domain, and an intracellular domain. Theextracellular domain contains a series of EGF-like repeats that areinvolved in ligand binding. During maturation, the Notch polypeptide iscleaved by a furin-like protease. This cleavage divides the Notchprotein into two subunits that are held together by noncovalentinteractions of the NRR. In the absence of ligand binding, the NRRdomain functions to keep the Notch protein in a protease-resistantconformation. The intracellular domain is a transcription factor calledNotch intracellular domain (NICD), which is released upon proteolyticcleavage by gamma secretase, in response to binding of the Notch proteinby a ligand. In mammals, the Notch ligands are Delta-like (e.g., DLL1and DLL4) and Jagged (also referred to as Jag, e.g., Jag1 and Jag2).When the NICD is released, it travels to the nucleus, where it activatestranscription of the Notch-responsive genes, HES1, HES5, NRARP, Deltex1and c-MYC. For reviews of Notch-related biology, see, e.g., Bray, 2006,NATURE REVIEWS 7:678-689; Kopan et al., 2009, CELL 137:216-233.

While Notch proteins play crucial roles in normal development,dysregulation of the Notch proteins is associated with various types ofcancer, including T-cell acute lymphatic leukemia/lymphoma (T-All),breast cancer, colon cancer, ovarian cancer and lung cancer. See, e.g.,Miele et al., 2006, CURRENT CANCER DRUG TARGETS 6:313-323. Accordingly,one therapeutic approach for the treatment of cancer is inhibition ofNotch pathway signaling. Inhibition of Notch pathway signaling has beenachieved using monoclonal antibodies (Wu et al., 2010, NATURE464:1052-1057; Aste-Amézaga et al., 2010, PLOS ONE 5:1-13 e9094).

Naturally-occurring antibodies are multimeric proteins that contain fourpolypeptide chains (FIG. 1). Two of the polypeptide chains are calledimmunoglobulin heavy chains (H chains), and two of the polypeptidechains are called immunoglobulin light chains (L chains). Theimmunoglobulin heavy and light chains are connected by an interchaindisulfide bond. The immunoglobulin heavy chains are connected byinterchain disulfide bonds. A light chain consists of one variableregion (V_(L) in FIG. 1) and one constant region (C_(L) in FIG. 1). Theheavy chain consists of one variable region (V_(H) in FIG. 1) and atleast three constant regions (CH₁, CH₂ and CH₃ in FIG. 1). The variableregions determine the specificity of the antibody.

Each variable region contains three hypervariable regions known ascomplementarity determining regions (CDRs) flanked by four relativelyconserved regions known as framework regions (FRs). The three CDRs,referred to as CDR₁, CDR₂, and CDR₃, contribute to the antibody bindingspecificity. Naturally occurring antibodies have been used as startingmaterial for engineered antibodies, such as chimeric antibodies andhumanized antibodies.

There is a need for improved antibodies that neutralize the biologicalactivity of human Notch3 and that can be used as therapeutic agents totreat human patients.

SUMMARY OF THE INVENTION

The invention is based on the discovery of antibodies that specificallybind human Notch3. Antibodies disclosed herein contain Notch3 bindingsites based on the CDRs of the anti-Notch3 antibodies described herein.The disclosed antibodies prevent or inhibit activation of human Notch3.They do so by inhibiting Notch3 from binding to Notch ligands, i.e.,Jag1, Jag2, DLL1, and DLL4. The disclosed antibodies can be used toinhibit the proliferation of tumor cells in vitro and/or in vivo. Whenadministered to a human cancer patient, the antibodies inhibit or reducetumor growth in the human patient.

These and other aspects and advantages of the invention are illustratedby the following figures, detailed description and claims. As usedherein, “including” means without limitation, and examples cited arenon-limiting. As used herein, “antibody 4F11” means antibody 4F11, orhumanized variants thereof.

DESCRIPTION OF THE DRAWINGS

The invention can be more completely understood with reference to thefollowing drawings.

FIG. 1 (prior art) is a schematic representation of a typicalnaturally-occurring antibody.

FIGS. 2A-C provide the amino acid sequence corresponding to theextracellular domain (ECD; amino acids 40 to 1643) of human Notch3 (SEQID NO:95). The amino acid sequence begins in FIG. 2A and continues inFIGS. 2B-C.

FIG. 3 provides the amino acid sequence corresponding to EGF-likerepeats 1-11 (amino acids 40 to 467 of the extracellular domain shown inFIGS. 2A-C) of human Notch3 (SEQ ID NO:96).

FIG. 4 is a bar chart summarizing results of an experiment to determinespecificity of antibody 4F11 binding to Fc-bound recombinant humanNotch3 (rhNotch3) using Octet. The results demonstrate that the bindingspecificity of the 4F11 antibody for rhNotch3 is much higher than forrhNotch1 or rhNotch2.

FIG. 5A is a bar chart illustrating the ability of antibody 4F11 toneutralize binding of the ligand Jag1 to human Notch3 protein. The chartshows that the antibody 4F11 and a Notch3 Specific Control, but nothuman immunoglobulin (hIgG), were able to block binding of recombinanthuman Fc-bound Jag1 (Jag1-Fc) to rhNotch3, as detected by bio-layerinterferometry (BLI).

FIG. 5B is a bar chart illustrating the ability of antibody 4F11 toneutralize binding of the ligand Jag2 to human Notch3 protein. The chartshows that the antibody 4F11 and a Notch3 Specific Control, but nothIgG, were able to block binding of recombinant human Fc-bound Jag2(Jag2-Fc) to rhNotch3, as detected by BLI.

FIG. 5C is a bar chart illustrating the ability of antibody 4F11 toneutralize binding of the ligand DLL1 to human Notch3 protein. The chartshows that the antibody 4F11 and a Notch3 Specific Control, but nothIgG, block binding of recombinant human Fc-bound DLL1 (DLL1-Fc) torhNotch3, to a greater extent than IgG, as detected by BLI.

FIG. 5D is a bar chart illustrating the ability of antibody 4F11 toneutralize binding of the ligand DLL4 to human Notch3 protein. The chartshows that the antibody 4F11 and a Notch3 Specific Control, but nothIgG, were able to block binding of recombinant human Fc-bound DLL4(DLL4-Fc) to rhNotch3, as detected by BLI.

FIG. 6A is a dose-response curve summarizing results from a Notch3reporter assay showing that antibody 4F11 inhibits Notch3-dependentreporter gene expression in the presence of the Fc-bound ligand Jag2(Jag2-Fc). The graph shows the relationship between inhibition ofJag2-Fc-stimulated reporter activity (% Inhibition) by antibody 4F11 intransduced cells, in relation to the amount of Jag2-Fc ligand. Reporteractivity of cells transduced with the RBP-Jκ-dependent luciferasereporter, exposed to any activating ligand, and treated with mouse IgG(mIgG) was defined as 100% activity for each ligand. Reporter activityof transduced cells not exposed to ligand but treated with mouse IgG wasdefined as 0% activity.

FIG. 6B is a bar chart summarizing results of Notch3 reporter assaysshowing that antibody 4F11, but not mouse IgG (mIgG), inhibitsNotch3-dependent reporter gene expression induced by the ligands hJag1,hJag2, hDLL1 and hDLL4. Reporter activity of cells transduced with theRBP-R-dependent luciferase reporter, exposed to any activating ligand,and treated with mouse IgG (mIgG) was defined as 100% activity for eachligand. Reporter activity of transduced cells not exposed to ligand buttreated with mouse IgG was defined as 0% activity.

FIG. 7 is a sequence alignment showing the amino acid sequence of thecomplete immunoglobulin heavy chain variable region of chimeric 4F11variable region denoted as Ch4F11 and humanized 4F11 heavy chainvariable regions denoted as Sh4F11 Hv3-23, Sh4F11 Hv3-23 A28T S31H T62S,Sh4F11 Hv3-23 S31H T62S, Sh4F11 Hv3-23 A28T S31N T62S, and Sh4F11 Hv3-23A28T T62S. The amino acid sequences for each heavy chain variable regionare aligned against one another, and CDR₁, CDR₂, and CDR₃ sequences(Kabat definition) are identified in boxes. The unboxed sequencesrepresent framework (FR) sequences.

FIG. 8 is a sequence alignment showing the isolated CDR₁, CDR₂, and CDR₃sequences for each of the immunoglobulin heavy chain variable regionsequences in FIG. 7.

FIG. 9 is a sequence alignment showing the amino acid sequence of thecomplete immunoglobulin light chain variable region of chimeric 4F11denoted as Ch4F11 and humanized 4F11 light chain variable regionsdenoted as Hu4F11 Kv2D-29, Hu4F11 Kv2D-29 N28H, Hu4F11 Kv2D-29 N28Q, andHu4F11 Kv2D-29 N28Y. The light chain variable region amino acidsequences are aligned against one another, and CDR₁, CDR₂, and CDR₃sequences (Kabat definition) are identified in boxes. The unboxedsequences represent framework (FR) sequences.

FIG. 10 is a sequence alignment showing the isolated CDR₁, CDR₂, andCDR₃ sequences for each of the immunoglobulin light chain variableregion sequences in FIG. 9.

FIG. 11 is a graph illustrating the ability of selected humanized 4F11(i.e., Hu4F11-70, Hu4F11-72, Hu4F11-74, and Hu4F11-78) antibodies toinhibit ligand-induced Notch3 ICD cleavage in MDA-MB-468 cells plated onJag2mFc. MDA-MB-468 cells exposed to either one of the 4F11 antibodiesor hIgG were plated in wells coated with hJag2-mFc, incubated overnight,washed, and their lysates collected for detection of NICD by Westernblot. The graph shows intensity of the cleaved NICD band in each sampleas detected by Western blot. Blots were also probed with anti-β tubulinas a control, and bands were quantitated using ImageLab software. As anegative control, cells exposed to hIgG were plated in plates coatedwith mFc (Fc) rather than Jag2mFc.

FIG. 12A and FIG. 12B are graphs summarizing results of a Notch3reporter assay showing that the humanized variants of 4F11 inhibitNotch3-dependent reporter gene expression induced by the ligand Jag2.CHO cells transfected with Jag2 cDNA expression vector were incubatedfor 24 hours with a mixture of HCC1143 reporter cells and 0.1 mg/ml, 1mg/ml, or 10 mg/ml of 4F11 antibody variants (i.e., Hu4F11-32,Hu4F11-69, Hu4F11-70, Hu4F11-71, Hu4F11-72, Hu4F11-73, Hu4F11-74,Hu4F11-75, Hu4F11-76, Hu4F11-77, Hu4F11-78, Hu4F11-79, or Hu4F11-80), anegative control antibody (Neg Control Ab), or human immunoglobulin G(IgG), or were left untreated. 24 hours after exposing reporter cells toJag2-expressing CHO cells, cells were processed using the Bright Glo(Promega, Madison, Wis.) reporter assay protocol and average relativelight units (RLU) were detected on a GloMax Luminometer (Promega) (FIG.12A and FIG. 12B).

FIG. 13 is a graph illustrating the ability of selected humanized 4F11antibodies (i.e., Hu4F11-70, Hu4F11-72 and Hu4F11-78) to inhibitactivation of Notch3 receptor in vivo after treatment of mice bearingHCC2429 xenograft tumors. Mice inoculated with matrigel and HCC2429Notch3-expressing cells were allowed to develop tumors of 300-400 mm³,at which point three mice (ml, m2, and m3 in FIG. 13) each were treatedwith 20 mg/kg of hIgG, 4F11, or humanized Notch3 antibody. Tumors werethen harvested and lysates collected, and Western blotting was performedusing an antibody specific for the Notch3 C-terminus in order to detectthe extent of Notch intracellular domain cleavage. Western blot bandintensity of the cleaved Notch intracellular domain fragment wascalculated, normalizing to β-tubulin loading control.

DETAILED DESCRIPTION

The antibodies disclosed herein are based on the antigen binding sitesof certain monoclonal antibodies that have been selected on the basis ofbinding and neutralizing the activity of human Notch3. The antibodiescontain immunoglobulin variable region CDR sequences that define abinding site for human Notch3.

Because of the neutralizing activity of these antibodies, they areuseful for inhibiting the growth and/or proliferation of certain cancercells and tumors. The antibodies can be engineered (e.g., humanized) tominimize or eliminate an immune response when administered as atherapeutic antibody to a human patient. Various features and aspects ofthe invention are discussed in more detail below.

As used herein, unless otherwise indicated, the term “antibody” means anintact antibody (e.g., an intact monoclonal antibody) or antigen-bindingfragment of an antibody, including an intact antibody or antigen-bindingfragment that has been modified, engineered or chemically conjugated.Examples of antibodies that have been modified or engineered arechimeric antibodies, humanized antibodies, and multispecific antibodies(e.g., bispecific antibodies). Examples of antigen-binding fragmentsinclude Fab, Fab′, F(ab′)₂, Fv, single chain antibodies (e.g., scFv),minibodies, and diabodies. An antibody conjugated to a toxin moiety isan example of a chemically conjugated antibody.

I. Antibodies that Bind Human Notch3

As disclosed herein, the antibodies may comprise: (a) an immunoglobulinheavy chain variable region comprising the structureCDR_(H1)-CDR_(H2)-CDR_(H3) and (b) an immunoglobulin light chainvariable region comprising the structure CDR_(L1)-CDR_(L2)-CDR_(L3),wherein the heavy chain variable region and the light chain variableregion together define a single binding site for binding human Notch3.

In some embodiments, the antibody comprises: (a) an immunoglobulin heavychain variable region comprising the structureCDR_(H1)-CDR_(H2)-CDR_(H3) and (b) an immunoglobulin light chainvariable region, wherein the heavy chain variable region and the lightchain variable region together define a single binding site for bindinghuman Notch3. A CDR_(H1) comprises an amino acid sequence selected fromthe group consisting of SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:14, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61; aCDR_(H2) comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:15 and SEQ ID NO:53;and a CDR_(H3) comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:7 and SEQ ID NO:16. (The immunoglobulin heavychain variable regions including the referenced CDR sequences can befound in Table 10 and FIGS. 7-8.)

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 5 and SEQ IDNO: 11, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO: 6,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 51 and SEQ IDNO: 54, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO: 53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 51 and SEQ IDNO: 55, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO: 53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 52 and SEQ IDNO: 56, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO: 53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 5 and SEQ IDNO: 57, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO: 53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO: 7.

Preferably, the CDR_(H1), CDR_(H2), and CDR_(H3) sequences areinterposed between human or humanized immunoglobulin FR sequences.

In other embodiments, the antibody comprises (a) an immunoglobulin lightchain variable region comprising the structureCDR_(L1)-CDR_(L2)-CDR_(L3), and (b) an immunoglobulin heavy chainvariable region, wherein the IgG light chain variable region and the IgGheavy chain variable region together define a single binding site forbinding human Notch3. A CDR_(L1) comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:8, SEQ ID NO:20, SEQ IDNO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 and SEQ IDNO:67; a CDR_(L2) comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO:9 or the amino acid sequence of KVS; and aCDR_(L3) comprises an amino acid sequence of SEQ ID NO:10. (Theimmunoglobulin light chain variable regions including the referenced CDRsequences can be found in Table 11 and FIGS. 9-10.)

In some embodiments, the antibody comprises an immunoglobulin lightchain variable region comprising a CDR_(L1) comprising the amino acidsequence of SEQ ID NO: 8, a CDR_(L2) comprising the amino acid sequenceof SEQ ID NO: 9, and a CDR_(L3) comprising the amino acid sequence ofSEQ ID NO: 10.

In some embodiments, the antibody comprises an immunoglobulin lightchain variable region comprising a CDR_(L1) comprising the amino acidsequence of SEQ ID NO: 62, a CDR_(L2) comprising the amino acid sequenceof SEQ ID NO: 9, and a CDR_(L3) comprising the amino acid sequence ofSEQ ID NO: 10.

In some embodiments, the antibody comprises an immunoglobulin lightchain variable region comprising a CDR_(L1) comprising the amino acidsequence of SEQ ID NO: 63, a CDR_(L2) comprising the amino acid sequenceof SEQ ID NO: 9, and a CDR_(L3) comprising the amino acid sequence ofSEQ ID NO: 10.

In some embodiments, the antibody comprises an immunoglobulin lightchain variable region comprising a CDR_(L1) comprising the amino acidsequence of SEQ ID NO: 64, a CDR_(L2) comprising the amino acid sequenceof SEQ ID NO: 9, and a CDR_(L3) comprising the amino acid sequence ofSEQ ID NO: 10.

Preferably, the CDR_(L1), CDR_(L2), and CDR_(L3) sequences areinterposed between human or humanized immunoglobulin FR sequences.

In some embodiments, the antibody comprises: (a) an immunoglobulin heavychain variable region comprising the structureCDR_(H1)-CDR_(H2)-CDR_(H3) and (b) an immunoglobulin light chainvariable region comprising the structure CDR_(L1)-CDR_(L2)-CDR_(L3),wherein the heavy chain variable region and the light chain variableregion together define a single binding site for binding human Notch3.The CDR_(H1) comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:51, SEQID NO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61; the CDR_(H2)comprises an amino acid sequence selected from the group consisting ofSEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:15 and SEQ ID NO:53; and theCDR_(H3) comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:7 and SEQ ID NO:16. The CDR_(L1) comprises anamino acid sequence selected from the group consisting of SEQ ID NO:8,SEQ ID NO:20, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65,SEQ ID NO:66 and SEQ ID NO:67; the CDR_(L2) comprises an amino acidsequence selected from the group consisting of SEQ ID NO:9 or the aminoacid sequence of KVS; and the CDR_(L3) comprises an amino acid sequenceof SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:5 and SEQ IDNO:11, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:6, anda CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:8, a CDR_(L2) comprisingthe amino acid sequence of SEQ ID NO:9, and a CDR_(L3) comprising theamino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 5 and SEQ IDNO:11; a CDR_(H2) comprising the amino acid sequence of SEQ ID NO: 6;and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO: 7; andan immunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO: 62; a CDR_(L2)comprising the amino acid sequence of SEQ ID NO: 9; and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 5 and SEQ IDNO:11; a CDR_(H2) comprising the amino acid sequence of SEQ ID NO: 6;and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO: 7; andan immunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO: 63; a CDR_(L2)comprising the amino acid sequence of SEQ ID NO: 9; and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 5 and SEQ IDNO:11; a CDR_(H2) comprising the amino acid sequence of SEQ ID NO: 6;and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO: 7; andan immunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO: 64; a CDR_(L2)comprising the amino acid sequence of SEQ ID NO: 9; and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:51 and SEQ IDNO: 54, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:8, a CDR_(L2) comprisingthe amino acid sequence of SEQ ID NO:9, and a CDR_(L3) comprising theamino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:51 and SEQ IDNO: 54, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:62, a CDR_(L2)comprising the amino acid sequence of SEQ ID NO:9, and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:51 and SEQ IDNO: 54, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:63, a CDR_(L2)comprising the amino acid sequence of SEQ ID NO:9, and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:51 and SEQ IDNO: 54, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:64, a CDR_(L2)comprising the amino acid sequence of SEQ ID NO:9, and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:51 and SEQ IDNO: 55, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:8, a CDR_(L2) comprisingthe amino acid sequence of SEQ ID NO:9, and a CDR_(L3) comprising theamino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:51 and SEQ IDNO: 55, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:62, a CDR_(L2)comprising the amino acid sequence of SEQ ID NO:9, and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:51 and SEQ IDNO: 55, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:63, a CDR_(L2)comprising the amino acid sequence of SEQ ID NO:9, and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:51 and SEQ IDNO: 55, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:64, a CDR_(L2)comprising the amino acid sequence of SEQ ID NO:9, and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:52 and SEQ IDNO: 56, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:8, a CDR_(L2) comprisingthe amino acid sequence of SEQ ID NO:9, and a CDR_(L3) comprising theamino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:52 and SEQ IDNO: 56, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:62, a CDR_(L2)comprising the amino acid sequence of SEQ ID NO:9, and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:52 and SEQ IDNO: 56, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:63, a CDR_(L2)comprising the amino acid sequence of SEQ ID NO:9, and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:52 and SEQ IDNO: 56, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:64, a CDR_(L2)comprising the amino acid sequence of SEQ ID NO:9, and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:5 and SEQ IDNO: 57, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:8, a CDR_(L2) comprisingthe amino acid sequence of SEQ ID NO:9, and a CDR_(L3) comprising theamino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:5 and SEQ IDNO: 57, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:62, a CDR_(L2)comprising the amino acid sequence of SEQ ID NO:9, and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:5 and SEQ IDNO: 57, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:63, a CDR_(L2)comprising the amino acid sequence of SEQ ID NO:9, and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO:10.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising a CDR_(H1) comprising an amino acidsequence selected from the group consisting of SEQ ID NO:5 and SEQ IDNO: 57, a CDR_(H2) comprising the amino acid sequence of SEQ ID NO:53,and a CDR_(H3) comprising the amino acid sequence of SEQ ID NO:7; and animmunoglobulin light chain variable region comprising a CDR_(L1)comprising the amino acid sequence of SEQ ID NO:64, a CDR_(L2)comprising the amino acid sequence of SEQ ID NO:9, and a CDR_(L3)comprising the amino acid sequence of SEQ ID NO:10.

The antibodies disclosed herein comprise an immunoglobulin heavy chainvariable region and an immunoglobulin light chain variable region. Insome embodiments, the antibody comprises an immunoglobulin heavy chainvariable region selected from the group consisting of SEQ ID NO:2 (4F11,Ch4F11 Chimeric); SEQ ID NO:34 (Sh4F11 Hv3-23); SEQ ID NO:36 (Sh4F11Hv3-23 A28T S31H T62S); SEQ ID NO:38 (Sh4F11 Hv3-23 S31H T62S); SEQ IDNO:40 (Sh4F11 Hv3-23 A28T S31N T62S); and SEQ ID NO:42 (Sh4F11 Hv3-23A28T T62S); and an immunoglobulin light chain variable region. As aconvenience for the reader, certain SEQ ID NOs. are followed by aparenthetical including the antibody designation that was the origin ofthe sequence. For example, “SEQ ID NO:2 (4F11, Ch4F11 Chimeric)” meansthat SEQ ID NO:2 comes from murine antibody 4F11. SEQ ID NO:2 is alsofound in the chimeric 4F11 sequence.

In other embodiments, the antibody comprises an immunoglobulin lightchain variable region selected from the group consisting of SEQ ID NO:4(4F11, Ch4F11 Chimeric); SEQ ID NO:44 (Hu4F11 Kv2D-29); SEQ ID NO:46(Hu4F11 Kv2D-29 N28H); SEQ ID NO:48 (Hu4F11 Kv2D-29 N28Q); and SEQ IDNO:50 (Hu4F11 Kv2D-29 N28Y); and an immunoglobulin heavy chain variableregion.

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region selected from the group consisting of SEQ ID NO:2(4F11, Ch4F11 Chimeric); SEQ ID NO:34 (Sh4F11 Hv3-23); SEQ ID NO:36(Sh4F11 Hv3-23 A28T S31H T62S); SEQ ID NO:38 (Sh4F11 Hv3-23 S31H T62S);SEQ ID NO:40 (Sh4F11 Hv3-23 A28T S31N T62S); and SEQ ID NO:42 (Sh4F11Hv3-23 A28T T62S), and an immunoglobulin light chain variable regionselected from the group consisting of SEQ ID NO:4 (4F11, Ch4F11Chimeric); SEQ ID NO:44 (Hu4F11 Kv2D-29); SEQ ID NO:46 (Hu4F11 Kv2D-29N28H); SEQ ID NO:48 (Hu4F11 Kv2D-29 N28Q); and SEQ ID NO:50 (Hu4F11Kv2D-29 N28Y).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:2(4F11, Ch4F11 Chimeric), and an immunoglobulin light chain variableregion comprising the amino acid sequence of SEQ ID NO:4 (4F11, Ch4F11Chimeric).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:2(4F11, Ch4F11 Chimeric), and an immunoglobulin light chain variableregion comprising the amino acid sequence of SEQ ID NO:44 (Hu4F11Kv2D-29).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:34(Sh4F11 Hv3-23), and an immunoglobulin light chain variable regioncomprising the amino acid sequence of SEQ ID NO:4 (4F11, Ch4F11Chimeric).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:34(Sh4F11 Hv3-23), and an immunoglobulin light chain variable regioncomprising the amino acid sequence of SEQ ID NO:44 (Hu4F11 Kv2D-29).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:36(Sh4F11 Hv3-23 A28T S31H T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:46(Hu4F11 Kv2D-29 N28H).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:38(Sh4F11 Hv3-23 S31H T62S), and an immunoglobulin light chain variableregion comprising the amino acid sequence of SEQ ID NO:46 (Hu4F11Kv2D-29 N28H).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:40(Sh4F11 Hv3-23 A28T S31N T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:46(Hu4F11 Kv2D-29 N28H).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:42(Sh4F11 Hv3-23 A28T T62S), and an immunoglobulin light chain variableregion comprising the amino acid sequence of SEQ ID NO:46 (Hu4F11Kv2D-29 N28H).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:36(Sh4F11 Hv3-23 A28T S31H T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:48(Hu4F11 Kv2D-29 N28Q).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:38(Sh4F11 Hv3-23 S31H T62S), and an immunoglobulin light chain variableregion comprising the amino acid sequence of SEQ ID NO:48 (Hu4F11Kv2D-29 N28Q).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:40(Sh4F11 Hv3-23 A28T S31N T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:48(Hu4F11 Kv2D-29 N28Q).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:42(Sh4F11 Hv3-23 A28T T62S), and an immunoglobulin light chain variableregion comprising the amino acid sequence of SEQ ID NO:48 (Hu4F11Kv2D-29 N28Q).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:36(Sh4F11 Hv3-23 A28T S31H T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:50(Hu4F11 Kv2D-29 N28Y).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:38(Sh4F11 Hv3-23 S31H T62S), and an immunoglobulin light chain variableregion comprising the amino acid sequence of SEQ ID NO:50 (Hu4F11Kv2D-29 N28Y).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:40(Sh4F11 Hv3-23 A28T S31N T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:50(Hu4F11 Kv2D-29 N28Y).

In some embodiments, the antibody comprises an immunoglobulin heavychain variable region comprising the amino acid sequence of SEQ ID NO:42(Sh4F11 Hv3-23 A28T T62S), and an immunoglobulin light chain variableregion comprising the amino acid sequence of SEQ ID NO:50 (Hu4F11Kv2D-29 N28Y).

In certain embodiments, the antibodies disclosed herein comprise animmunoglobulin heavy chain and an immunoglobulin light chain. In someembodiments, the antibody comprises an immunoglobulin heavy chainselected from the group consisting of SEQ ID NO: 27 (4F11); SEQ ID NO:74(Ch4F11 Chimeric); SEQ ID NO:76 (Sh4F11 Hv3-23); SEQ ID NO:78 (Sh4F11Hv3-23 A28T S31H T62S); SEQ ID NO:80 (Sh4F11 Hv3-23 S31H T62S); SEQ IDNO:82 (Sh4F11 Hv3-23 A28T S31N T62S); and SEQ ID NO:84 (Sh4F11 Hv3-23A28T T62S); and an immunoglobulin light chain.

In other embodiments, the antibody comprises an immunoglobulin lightchain selected from the group consisting of SEQ ID NO:29 (4F11); SEQ IDNO:86 (Ch4F11 Chimeric); SEQ ID NO:88 (Hu4F11 Kv2D-29 Kappa); SEQ IDNO:90 (Hu4F11 Kv2D-29 N28H Kappa); SEQ ID NO:92 (Hu4F11 Kv2D-29 N28QKappa); and SEQ ID NO:94 (Hu4F11 Kv2D-29 N28Y Kappa); and animmunoglobulin heavy chain.

In some embodiments, the antibody comprises an immunoglobulin heavychain selected from the group consisting of SEQ ID NO: 27 (4F11); SEQ IDNO:74 (Ch4F11 Chimeric); SEQ ID NO:76 (Sh4F11 Hv3-23); SEQ ID NO:78(Sh4F11 Hv3-23 A28T S31H T62S); SEQ ID NO:80 (Sh4F11 Hv3-23 S31H T62S);SEQ ID NO:82 (Sh4F11 Hv3-23 A28T S31N T62S); and SEQ ID NO:84 (Sh4F11Hv3-23 A28T T62S); and an immunoglobulin light chain selected from thegroup consisting of SEQ ID NO:29 (4F11); SEQ ID NO:86 (Ch4F11 Chimeric);SEQ ID NO:88 (Hu4F11 Kv2D-29 Kappa); SEQ ID NO:90 (Hu4F11 Kv2D-29 N28HKappa); SEQ ID NO:92 (Hu4F11 Kv2D-29 N28Q Kappa); and SEQ ID NO:94(Hu4F11 Kv2D-29 N28Y Kappa).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 27 (4F11), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 29 (4F11).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 74 (Ch4F11), andan immunoglobulin light chain comprising the amino acid sequence of SEQID NO: 86 (Ch4F11).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 74 (Ch4F11), andan immunoglobulin light chain comprising the amino acid sequence of SEQID NO: 88 (Hu4F11 Kv2D-29).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 76 (Sh4F11Hv3-23), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 86 (Ch4F11).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 76 (Sh4F11Hv3-23), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 88 (Hu4F11 Kv2D-29).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 78 (Sh4F11 Hv3-23A28T S31H T62S), and an immunoglobulin light chain comprising the aminoacid sequence of SEQ ID NO: 90 (Hu4F11 Kv2D-29 N28H Kappa).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 80 (Sh4F11 Hv3-23S31H T62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 90 (Hu4F11 Kv2D-29 N28H Kappa).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 82 (Sh4F11 Hv3-23A28T S31N T62S), and an immunoglobulin light chain comprising the aminoacid sequence of SEQ ID NO: 90 (Hu4F11 Kv2D-29 N28H Kappa).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 84 (Sh4F11 Hv3-23A28T T62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 90 (Hu4F11 Kv2D-29 N28H Kappa).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 78 (Sh4F11 Hv3-23A28T S31H T62S), and an immunoglobulin light chain comprising the aminoacid sequence of SEQ ID NO: 92 (Hu4F11 Kv2D-29 N28Q Kappa).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 80 (Sh4F11 Hv3-23S31H T62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 92 (Hu4F11 Kv2D-29 N28Q Kappa).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 82 (Sh4F11 Hv3-23A28T S31N T62S), and an immunoglobulin light chain comprising the aminoacid sequence of SEQ ID NO: 92 (Hu4F11 Kv2D-29 N28Q Kappa).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 84 (Sh4F11 Hv3-23A28T T62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 92 (Hu4F11 Kv2D-29 N28Q Kappa).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 78 (Sh4F11 Hv3-23A28T S31H T62S), and an immunoglobulin light chain comprising the aminoacid sequence of SEQ ID NO: 94 (Hu4F11 Kv2D-29 N28Y Kappa).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 80 (Sh4F11 Hv3-23S31H T62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 94 (Hu4F11 Kv2D-29 N28Y Kappa).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 82 (Sh4F11 Hv3-23A28T S31N T62S), and an immunoglobulin light chain comprising the aminoacid sequence of SEQ ID NO: 94 (Hu4F11 Kv2D-29 N28Y Kappa).

In some embodiments, the antibody comprises an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 84 (Sh4F11 Hv3-23A28T T62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 94 (Hu4F11 Kv2D-29 N28Y Kappa).

In other embodiments, an isolated antibody that binds human Notch3comprises an immunoglobulin heavy chain variable region comprising anamino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%,or 99% identical to the entire variable region or the FR sequence of SEQID NO:2 (4F11, Ch4F11 Chimeric); SEQ ID NO:34 (Sh4F11 Hv3-23); SEQ IDNO:36 (Sh4F11 Hv3-23 A28T S31H T62S); SEQ ID NO:38 (Sh4F11 Hv3-23 S31HT62S); SEQ ID NO:40 (Sh4F11 Hv3-23 A28T S31N T62S); or SEQ ID NO:42(Sh4F11 Hv3-23 A28T T62S).

In other embodiments, an isolated antibody that binds human Notch3comprises an immunoglobulin light chain variable region comprising anamino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%,or 99% identical to the entire variable region or the FR sequence of SEQID NO:4 (4F11, Ch4F11 Chimeric); SEQ ID NO:44 (Hu4F11 Kv2D-29); SEQ IDNO:46 (Hu4F11 Kv2D-29 N28H); SEQ ID NO:48 (Hu4F11 Kv2D-29 N28Q); or SEQID NO:50 (Hu4F11 Kv2D-29 N28Y).

Sequence identity may be determined in various ways that are within theskill of a person skilled in the art, e.g., using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. BLAST (Basic Local Alignment Search Tool) analysis using thealgorithm employed by the programs blastp, blastn, blastx, tblastn andtblastx (Karlin et al., (1990) PROC. NATL. ACAD. Sci. USA 87:2264-2268;Altschul, (1993) J. MOL. EVOL. 36:290-300; Altschul et al., (1997)NUCLEIC ACIDS RES. 25:3389-3402, herein incorporated by reference) aretailored for sequence similarity searching. For a discussion of basicissues in searching sequence databases see Altschul et al., (1994)NATURE GENETICS 6:119-129, herein fully incorporated by reference. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full length of the sequences being compared. The searchparameters for histogram, descriptions, alignments, expect (i.e., thestatistical significance threshold for reporting matches againstdatabase sequences), cutoff, matrix and filter are at the defaultsettings. The default scoring matrix used by blastp, blastx, tblastn,and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL.ACAD. SCI. USA 89:10915-10919, fully incorporated by reference herein).Four blastn parameters may be adjusted as follows: Q=10 (gap creationpenalty); R=10 (gap extension penalty); wink=1 (generates word hits atevery wink.sup.th position along the query); and gapw=16 (sets thewindow width within which gapped alignments are generated). Theequivalent Blastp parameter settings may be Q=9; R=2; wink=1; andgapw=32. Searches may also be conducted using the NCBI (National Centerfor Biotechnology Information) BLAST Advanced Option parameter (e.g.:−G, Cost to open gap [Integer]: default=5 for nucleotides/11 forproteins; −E, Cost to extend gap [Integer]: default=2 for nucleotides/1for proteins; −q, Penalty for nucleotide mismatch [Integer]: default=−3;−r, reward for nucleotide match [Integer]: default=1; −e, expect value[Real]: default=10; −W, wordsize [Integer]: default=11 fornucleotides/28 for megablast/3 for proteins; −y, Dropoff (X) for blastextensions in bits: default=20 for blastn/7 for others; −X, X dropoffvalue for gapped alignment (in bits): default=15 for all programs, notapplicable to blastn; and −Z, final X dropoff value for gapped alignment(in bits): 50 for blastn, 25 for others). ClustalW for pairwise proteinalignments may also be used (default parameters may include, e.g.,Blosum62 matrix and Gap Opening Penalty=10 and Gap ExtensionPenalty=0.1). A Bestfit comparison between sequences, available in theGCG package version 10.0, uses DNA parameters GAP=50 (gap creationpenalty) and LEN=3 (gap extension penalty) and the equivalent settingsin protein comparisons are GAP=8 and LEN=2.

In each of the foregoing embodiments, it is contemplated herein thatimmunoglobulin heavy chain variable region sequences and/or light chainvariable region sequences that together bind human Notch3 may containamino acid alterations (e.g., at least 1, 2, 3, 4, 5, or 10 amino acidsubstitutions, deletions, or additions) in the framework regions of theheavy and/or light chain variable regions.

In certain embodiments, an isolated antibody binds human Notch3 with aK_(D) of about 35 nM, 25 nM, 15 nM 10 nM, 5 nM, 1 nM, 900 pM, 850 pM,800 pM, 750 pM, 700 pM, 650 pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM,200 pM or less. Unless otherwise specified, K_(D) values are determinedby surface plasmon resonance methods under the conditions described inExamples 3 and 9.

In some embodiments, monoclonal antibodies bind to the same epitope onhuman Notch3 as antibody 4F11. In some embodiments, monoclonalantibodies compete for binding to human Notch3 with antibody 4F11. Forexample, monoclonal antibodies may compete for binding to theextracellular domain (ECD) of Notch3 with antibody 4F11 (amino acidsequence corresponding to the human Notch3 ECD is shown in FIGS. 2A-C).In another example, monoclonal antibodies may compete for binding toEGF-like repeats 1-11 of human Notch3 with antibody 4F11 (amino acidsequence corresponding to EGF-like repeats 1-11 of human Notch3 is shownin FIG. 3).

Competition assays for determining whether an antibody binds to the sameepitope as, or competes for binding with, antibody 4F11 are known in theart. Exemplary competition assays include immunoassays (e.g., ELISAassays, RIA assays), BIAcore analysis, biolayer interferometry and flowcytometry.

Typically, a competition assay involves the use of an antigen (e.g., ahuman Notch3 protein or fragment thereof) bound to a solid surface orexpressed on a cell surface, a test anti-Notch3-binding antibody and areference antibody (i.e., antibody 4F11). The reference antibody islabeled and the test antibody is unlabeled. Competitive inhibition ismeasured by determining the amount of labeled reference antibody boundto the solid surface or cells in the presence of the test antibody.Usually the test antibody is present in excess (e.g., 1×, 5×, 10×, 20×or 100×). Antibodies identified by competition assay (i.e., competingantibodies) include antibodies binding to the same epitope, or similar(e.g., overlapping) epitopes, as the reference antibody, and antibodiesbinding to an adjacent epitope sufficiently proximal to the epitopebound by the reference antibody for steric hindrance to occur.

In an exemplary competition assay, a reference anti-Notch3 antibody(i.e., antibody 4F11) is biotinylated using commercially availablereagents. The biotinylated reference antibody is mixed with serialdilutions of the test antibody or unlabeled reference antibody(self-competition control) resulting in a mixture of various molarratios (e.g., 1×, 5×, 10×, 20× or 100×) of test antibody (or unlabeledreference antibody) to labeled reference antibody. The antibody mixtureis added to a human Notch3 (e.g., extracellular domain of human Notch3)polypeptide coated-ELISA plate. The plate is then washed and horseradishperoxidase (HRP)-strepavidin is added to the plate as the detectionreagent. The amount of labeled reference antibody bound to the targetantigen is detected following addition of a chromogenic substrate (e.g.,TMB (3,3′,5,5′-tetramethylbenzidine) or ABTS(2,2″-azino-di-(3-ethylbenzthiazoline-6-sulfonate)), which are known inthe art. Optical density readings (OD units) are measured using aSpectraMax® M2 spectrometer (Molecular Devices). OD units correspondingto zero percent inhibition are determined from wells without anycompeting antibody. OD units corresponding to 100% inhibition, i.e., theassay background are determined from wells without any labeled referenceantibody or test antibody. Percent inhibition of labeled referenceantibody to Notch3 by the test antibody (or the unlabeled referenceantibody) at each concentration is calculated as follows: %inhibition=(1−(OD units−100% inhibition)/(0% inhibition−100%inhibition))*100. Persons skilled in the art will appreciate that thecompetition assay can be performed using various detection systems knownin the art.

A competition assay may be conducted in both directions to ensure thatthe presence of the label does not interfere or otherwise inhibitbinding. For example, in the first direction the reference antibody islabeled and the test antibody is unlabeled, and in the second direction,the test antibody is labeled and the reference antibody is unlabeled.

A test antibody competes with the reference antibody for specificbinding to the antigen if an excess of one antibody (e.g., 1×, 5×, 10×,20× or 100×) inhibits binding of the other antibody, e.g., by at least50%, 75%, 90%, 95% or 99% as measured in a competitive binding assay.

Two antibodies may be determined to bind to the same epitope ifessentially all amino acid mutations in the antigen that reduce oreliminate binding of one antibody reduce or eliminate binding of theother. Two antibodies may be determined to bind to overlapping epitopesif only a subset of the amino acid mutations that reduce or eliminatebinding of antibody reduce or eliminate binding of the other.

II. Antibody Production

Methods for producing antibodies of the invention are known in the art.For example, DNA molecules encoding light chain variable regions and/orheavy chain variable regions can be chemically synthesized using thesequence information provided herein. Synthetic DNA molecules can beligated to other appropriate nucleotide sequences, including, e.g.,constant region coding sequences, and expression control sequences, toproduce conventional gene expression constructs encoding the desiredantibody. Production of defined gene constructs is within routine skillin the art. Alternatively, the sequences provided herein can be clonedout of hybridomas by conventional hybridization techniques or polymerasechain reaction (PCR) techniques, using synthetic nucleic acid probeswhose sequences are based on sequence information provided herein, orprior art sequence information regarding genes encoding the heavy andlight chains of murine antibodies in hybridoma cells.

Nucleic acids encoding desired antibodies can be incorporated (ligated)into expression vectors, which can be introduced into host cells throughconventional transfection or transformation techniques. Exemplary hostcells are E. coli cells, Chinese hamster ovary (CHO) cells, HeLa cells,baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells that donot otherwise produce IgG protein. Transformed host cells can be grownunder conditions that permit the host cells to express the genes thatencode the immunoglobulin light and/or heavy chain variable regions.

Specific expression and purification conditions will vary depending uponthe expression system employed. For example, if a gene is to beexpressed in E. coli, it is first cloned into an expression vector bypositioning the engineered gene downstream from a suitable bacterialpromoter, e.g., Trp or Tac, and a prokaryotic signal sequence. Theexpressed secreted protein accumulates in refractile or inclusionbodies, and can be harvested after disruption of the cells by Frenchpress or sonication. The refractile bodies then are solubilized, and theproteins refolded and cleaved by methods known in the art.

If the engineered gene is to be expressed in eukaryotic host cells,e.g., CHO cells, it is first inserted into an expression vectorcontaining a suitable eukaryotic promoter, a secretion signal, a poly Asequence, and a stop codon, and, optionally, may contain enhancers, andvarious introns. This expression vector optionally contains sequencesencoding all or part of a constant region, enabling an entire, or a partof, a heavy or light chain to be expressed. The gene construct can beintroduced into eukaryotic host cells using conventional techniques. Thehost cells express V_(L) or V_(H) fragments, V_(L)-V_(H) heterodimers,V_(H)-V_(L) or V_(L)-V_(H) single chain polypeptides, complete heavy orlight immunoglobulin chains, or portions thereof, each of which may beattached to a moiety having another function (e.g., cytotoxicity). Insome embodiments, a host cell is transfected with a single vectorexpressing a polypeptide expressing an entire, or part of, a heavy chain(e.g., a heavy chain variable region) or a light chain (e.g., a lightchain variable region). In other embodiments, a host cell is transfectedwith a single vector encoding (a) a polypeptide comprising a heavy chainvariable region and a polypeptide comprising a light chain variableregion, or (b) an entire immunoglobulin heavy chain and an entireimmunoglobulin light chain. In still other embodiments, a host cell isco-transfected with more than one expression vector (e.g., oneexpression vector encoding a polypeptide comprising an entire, or partof, a heavy chain or heavy chain variable region, and another expressionvector encoding a polypeptide comprising an entire, or part of, a lightchain or light chain variable region).

A polypeptide comprising an immunoglobulin heavy chain variable regionor light chain variable region can be produced by growing (culturing) ahost cell transfected with an expression vector encoding such variableregion, under conditions that permit expression of the polypeptide.Following expression, the polypeptide can be harvested and purified orisolated using techniques known in the art, e.g., affinity tags such asglutathione-S-transferase (GST) and histidine tags.

A monoclonal antibody that binds human Notch3, or an antigen-bindingfragment of the antibody, can be produced by growing (culturing) a hostcell transfected with: (a) an expression vector that encodes a completeor partial immunoglobulin heavy chain, and a separate expression vectorthat encodes a complete or partial immunoglobulin light chain; or (b) asingle expression vector that encodes both chains (e.g., complete orpartial heavy and light chains), under conditions that permit expressionof both chains. The intact antibody (or antigen-binding fragment of theantibody) can be harvested and purified or isolated using techniquesknown in the art, e.g., Protein A, Protein G, affinity tags such asglutathione-S-transferase (GST) and histidine tags. It is withinordinary skill in the art to express the heavy chain and the light chainfrom a single expression vector or from two separate expression vectors.

III. Antibody Modifications

Methods for reducing or eliminating the antigenicity of antibodies andantibody fragments are known in the art. When administered to a human,the disclosed antibodies preferably are “humanized” to reduce oreliminate antigenicity in humans. Preferably, the humanized antibodieshave the same, or substantially the same, affinity for the antigen asthe non-humanized mouse antibody from which it was derived.

In one humanization approach, chimeric proteins are created in whichmouse immunoglobulin constant regions are replaced with humanimmunoglobulin constant regions. See, e.g., Morrison et al., 1984, PROC.NAT. ACAD. CI. 81:6851-6855, Neuberger et al., 1984, NATURE 312:604-608;U.S. Pat. No. 6,893,625 (Robinson); U.S. Pat. No. 5,500,362 (Robinson);and U.S. Pat. No. 4,816,567 (Cabilly).

In an approach known as CDR grafting, the CDRs of the light and heavychain variable regions are grafted into frameworks from another species.For example, murine CDRs can be grafted into human FRs. In someembodiments of the invention, the CDRs of the light and heavy chainvariable regions of an anti-Notch3 antibody are grafted into human FRsor consensus human FRs. To create consensus human FRs, FRs from severalhuman heavy chain or light chain amino acid sequences are aligned toidentify a consensus amino acid sequence. CDR grafting is described inU.S. Pat. No. 7,022,500 (Queen); U.S. Pat. No. 6,982,321 (Winter); U.S.Pat. No. 6,180,370 (Queen); U.S. Pat. No. 6,054,297 (Carter); U.S. Pat.No. 5,693,762 (Queen); U.S. Pat. No. 5,859,205 (Adair); U.S. Pat. No.5,693,761 (Queen); U.S. Pat. No. 5,565,332 (Hoogenboom); U.S. Pat. No.5,585,089 (Queen); U.S. Pat. No. 5,530,101 (Queen); Jones et al. (1986)NATURE 321: 522-525; Riechmann et al. (1988) NATURE 332: 323-327;Verhoeyen et al. (1988) SCIENCE 239: 1534-1536; and Winter (1998) FEBSLETT 430: 92-94.

In an approach called “SUPERHUMANIZATION™,” human CDR sequences arechosen from human germline genes, based on the structural similarity ofthe human CDRs to those of the mouse antibody to be humanized See, e.g.,U.S. Pat. No. 6,881,557 (Foote); and Tan et al., 2002, J. IMMUNOL169:1119-1125.

Other methods to reduce immunogenicity include “reshaping,”“hyperchimerization,” and “veneering/resurfacing.” See, e.g., Vaswami etal., 1998, ANNALS OF ALLERGY, ASTHMA, & IMMUNOL. 81:105; Roguska et al.,1996, PROT. ENGINEER 9:895-904; and U.S. Pat. No. 6,072,035 (Hardman) Inthe veneering/resurfacing approach, the surface accessible amino acidresidues in the murine antibody are replaced by amino acid residues morefrequently found at the same positions in a human antibody. This type ofantibody resurfacing is described, e.g., in U.S. Pat. No. 5,639,641(Pedersen).

Another approach for converting a mouse antibody into a form suitablefor medical use in humans is known as ACTIVMAB™ technology (Vaccinex,Inc., Rochester, N.Y.), which involves a vaccinia virus-based vector toexpress antibodies in mammalian cells. High levels of combinatorialdiversity of IgG heavy and light chains are said to be produced. See,e.g., U.S. Pat. No. 6,706,477 (Zauderer); U.S. Pat. No. 6,800,442(Zauderer); and U.S. Pat. No. 6,872,518 (Zauderer).

Another approach for converting a mouse antibody into a form suitablefor use in humans is technology practiced commercially by KaloBiosPharmaceuticals, Inc. (Palo Alto, Calif.). This technology involves theuse of a proprietary human “acceptor” library to produce an “epitopefocused” library for antibody selection.

Another approach for modifying a mouse antibody into a form suitable formedical use in humans is HUMAN ENGINEERING™ technology, which ispracticed commercially by XOMA (US) LLC. See, e.g., PCT Publication No.WO 93/11794 and U.S. Pat. No. 5,766,886 (Studnicka); U.S. Pat. No.5,770,196 (Studnicka); U.S. Pat. No. 5,821,123 (Studnicka); and U.S.Pat. No. 5,869,619 (Studnicka).

Any suitable approach, including any of the above approaches, can beused to reduce or eliminate human immunogenicity of an antibody of theinvention.

The antibody can be conjugated to an effector moiety such as a smallmolecule toxin or a radionuclide using standard in vitro conjugationchemistries. If the effector moiety is a polypeptide, the antibody canbe chemically conjugated to the effector or joined to the effector as afusion protein. Construction of fusion proteins is within ordinary skillin the art.

IV. Use of Antibodies

Antibodies disclosed herein may be engineered (e.g., humanized) foradministration to humans. Antibodies disclosed herein can be used totreat various forms of cancer, e.g., breast, ovarian, prostate,cervical, colorectal, lung, pancreatic, gastric, and head and neckcancers. The cancer cells are exposed to a therapeutically effectiveamount of the antibody so as to inhibit or reduce proliferation of thecancer cells. In some embodiments, the antibodies inhibit cancer cellproliferation by at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or100%.

In some embodiments, the disclosed antibodies may inhibit or reduceproliferation of a tumor cell by inhibiting binding of human Notch3 to aligand, e.g., Jag1, Jag2, DLL1, and DLL4. The disclosed antibodies canbe used in a method to inhibit tumor growth in a human patient. Themethod comprises administering to the patient a therapeuticallyeffective amount of the antibody.

Cancers associated with Notch3 overexpression and/or activation include,but are not limited to, breast cancer, ovarian cancer, prostate cancer,cervical cancer, lung cancer, brain cancer (e.g., glioblastoma,astrocytoma, neuroblastoma), melanomas, gastrointestinal cancers (e.g.,colorectal, pancreatic, and gastric), head and neck cancer, sarcomas(e.g. rhabdomyosarcoma, osteosarcoma), and hematopoietic cell cancers,(e.g., multiple myeloma, leukemia, e.g., precursor T acute lymphoblasticleukemia (T-ALL), precursor B acute lymphoblastic leukemia (B-ALL) andB-cell chronic lymphoblastic leukemia (B-CLL)).

As used herein, “treat”, “treating” and “treatment” mean the treatmentof a disease in a mammal, e.g., in a human. This includes: (a)inhibiting the disease, i.e., arresting its development; and (b)relieving the disease, i.e., causing regression of the disease state.

Generally, a therapeutically effective amount of active component is inthe range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1 mg/kgto 10 mg/kg. The amount administered will depend on variables such asthe type and extent of disease or indication to be treated, the overallhealth of the patient, the in vivo potency of the antibody, thepharmaceutical formulation, and the route of administration. The initialdosage can be increased beyond the upper level in order to rapidlyachieve the desired blood-level or tissue-level. Alternatively, theinitial dosage can be smaller than the optimum, and the daily dosage maybe progressively increased during the course of treatment. Human dosagecan be optimized, e.g., in a conventional Phase I dose escalation studydesigned to run from 0.5 mg/kg to 20 mg/kg. Dosing frequency can vary,depending on factors such as route of administration, dosage amount,serum half-life of the antibody, and the disease being treated.Exemplary dosing frequencies are once per day, once per week and onceevery two weeks. A preferred route of administration is parenteral,e.g., intravenous infusion. Formulation of monoclonal antibody-baseddrugs is within ordinary skill in the art. In some embodiments, amonoclonal antibody is lyophilized, and then reconstituted in bufferedsaline, at the time of administration.

For therapeutic use, an antibody preferably is combined with apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” means buffers, carriers, and excipients suitable foruse in contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio. Thecarrier(s) should be “acceptable” in the sense of being compatible withthe other ingredients of the formulations and not deleterious to therecipient. Pharmaceutically acceptable carriers include buffers,solvents, dispersion media, coatings, isotonic and absorption delayingagents, and the like, that are compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is known in the art.

Pharmaceutical compositions containing antibodies disclosed herein canbe presented in a dosage unit form and can be prepared by any suitablemethod. A pharmaceutical composition should be formulated to becompatible with its intended route of administration. Examples of routesof administration are intravenous (IV), intradermal, inhalation,transdermal, topical, transmucosal, and rectal administration. Apreferred route of administration for monoclonal antibodies is IVinfusion. Useful formulations can be prepared by methods known in thepharmaceutical art. For example, see Remington's PharmaceuticalSciences, 18th ed. (Mack Publishing Company, 1990). Formulationcomponents suitable for parenteral administration include a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as EDTA; buffers such as acetates, citrates orphosphates; and agents for the adjustment of tonicity such as sodiumchloride or dextrose.

For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). The carrier should be stable under theconditions of manufacture and storage, and should be preserved againstmicroorganisms. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol), and suitablemixtures thereof.

Pharmaceutical formulations preferably are sterile. Sterilization can beaccomplished by any suitable method, e.g., filtration through sterilefiltration membranes. Where the composition is lyophilized, filtersterilization can be conducted prior to or following lyophilization andreconstitution.

EXAMPLES

The following Examples are merely illustrative and are not intended tolimit the scope or content of the invention in any way.

Example 1: Production of Anti-hNotch3 Monoclonal Antibodies

Immunizations, fusions, and primary screens were conducted usingconventional methods following the Repetitive Immunization MultipleSites (RIMMS) protocol. Five AJ mice and five Balb/c mice were immunizedwith a protein containing amino acids 1-428 of human Notch3 fused to theFc portion of human IgG. In addition, five AJ and five Balb/c mice wereimmunized with a concatemeric protein containing two repeats of theregion of Notch3 comprising EGF-like domains 9-12. From eachimmunization strategy, two AJ mice and 2 Balb/c mice having seradisplaying high binding to immunogen by Enzyme Linked ImmunoSorbentAssay (ELISA) were chosen for subsequent fusion. Spleens and lymph nodesfrom the selected mice were harvested. B cells were harvested and fusedwith a myeloma line. Fusion products from AJ mice and Balb/c mice wereserially diluted in forty 96-well plates to near clonality. A total of5,280 supernatants from the cell fusions were screened for binding tohuman Notch3 on the surface of CHO cells, using a Mesoscaleelectrochemiluminescence assay (MSD). In total, four hundred and twentysupernatants that bound human Notch3 in this assay were identified fromthe AJ and Balb/c fusions. These fusion products were furthercharacterized by in vitro biochemical and cell-based assays, asdiscussed below. A panel of hybridomas was selected, the hybridomas weresubcloned, and monoclonal hybridomas were expanded. Antibodies wereexpressed from the hybridoma cell lines and purified by affinitychromatography on Protein G resin under standard conditions.

Example 2: Sequence Analysis of Anti-Notch3 Monoclonal Antibodies

The light chain isotype and heavy chain isotype of the monoclonalantibody, 4F11, in Example 1 was determined using the IsoStrip™ MouseMonoclonal Antibody Isotyping Kit according to the manufacturer'sinstructions (Roche Applied Science, Indianapolis, Ind.). The antibodywas determined to be kappa light chain and IgG1 heavy chain.

The heavy and light chain variable regions of the mouse monoclonalantibody were sequenced using 5′ RACE (Rapid Amplification of cDNAEnds). Total RNA was extracted from the hybridoma cell line using theRNeasy Miniprep kit according to the vendor's instructions (Qiagen,Valencia, Calif.). Full-length first strand cDNA containing 5′ ends wasgenerated using the SMARTer™ RACE cDNA Amplification Kit (Clontech,Mountain View, Calif.) according to the manufacturer's instructionsusing random primers for 5′ RACE.

The variable regions of the light (kappa) and heavy (IgG1) chains wereamplified by PCR, using KOD Hot Start Polymerase (EMD Chemicals,Gibbstown, N.J.) according to the manufacturer's instructions. Foramplification of 5′ cDNA ends in conjunction with the SMARTer™ RACE cDNAAmplification Kit, the Universal Primer Mix A primer (Clontech), a mixof 5′CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT 3′ (SEQ ID NO: 13)and 5′ CTAATACGACTCACTATAGGGC 3′ (SEQ ID NO: 17), was used as a 5′primer. The heavy chain variable region was amplified using the above 5′primers and a 3′ IgG1 constant region specific primer, 5′TATGCAAGGCTTACAACCACA 3′ (SEQ ID NO: 18). The kappa chain variableregion was amplified with the above 5′ primers and a 3′ kappa constantregion specific primer, 5′ CGACTGAGGCACCTCCAGATGTT 3′ (SEQ ID NO: 19).

Individual PCR products were isolated by agarose gel electrophoresis andpurified using the Qiaquick Gel Purification kit according to themanufacturer's instructions (Qiagen). The PCR products were subsequentlycloned into the pCR®4Blunt using the Zero Blunt® TOPO® PCR Cloning Kitaccording to the manufacturer's instructions (Invitrogen) andtransformed into DH5-α bacteria (Invitrogen) through standard molecularbiology techniques. Plasmid DNA isolated from transformed bacterialclones was sequenced using M13 Forward (5′ GTAAAACGACGGCCAGT 3′) (SEQ IDNO: 21) and M13 Reverse primers (5′ CAGGAAACAGCTATGACC 3′) (SEQ ID NO:30) by Beckman Genomics (Danvers, Mass.), using standard dideoxy DNAsequencing methods to identify the sequence of the variable regionsequences. The sequences were analyzed using Vector NTI software(Invitrogen) and the IMGT/V-Quest web server (available on the worldwide web at imgt.cines.fr) to identify and confirm variable regionsequences.

The nucleic acid sequences encoding and the protein sequences definingvariable regions of the murine monoclonal antibodies are summarizedbelow (amino terminal signal peptide sequences are not shown). CDRsequences (Kabat definition) are shown in bold and are underlined in theamino acid sequences.

Nucleic Acid Sequence Encoding the Heavy Chain Variable Region of the4F11 Antibody (SEQ ID NO: 1)

  1 gaagtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 61 tcctgtgcag cctctggatt cgctttcagt agctatgaca tgtcttgggt tcgccagact121 ccggagaaga ggctggagtg ggtcgcatac attagtcgtg gtggtggtag cacctactat181 ccagacactg tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgtac241 ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgg aagacatgct301 actacggcct actggtactt cgatgtctgg ggcgcaggga ccacggtcac cgtctcctca

Protein Sequence Defining the Heavy Chain Variable Region of the 4F11Antibody (SEQ ID NO: 2)

 1 evqlvesggg lvkpggslkl scaasgfafs  sydms wvrqt pekrlewva y  isrgggstyy 61 pdtvkg rfti srdnakntly lqmss1ksed tamyycgr ha   ttaywyfdvw gagttvtvss

Nucleic Acid Sequence Encoding the Kappa Chain Variable Region of the4F11 Antibody (SEQ ID NO: 3)

  1 gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 61 atctcttgca gatctagtca gagccttgta cacactaatg gcaacaccta tttacattgg121 tacctgcaga agccaggcca gtctccaaaa ctcctgatct acaaagtttc caaccgattt181 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc241 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg301 tggacgttcg gtggaggcac caagctggaa atcaaa

Protein Sequence Defining the Kappa Chain Variable Region of the 4F11Antibody (SEQ ID NO: 4)

 1 dvvmtqtpls lpvslgdqas isc rssqslv   htngntylh w ylqkpgqspk lliykvsnrf 61  s gvpdrfsgs gsgtdftlkl srveaedlgv yfc sqsthvp   wtfgggtkle ik

Table 1 is a concordance chart showing the SEQ ID NO. of each sequencediscussed in this Example.

TABLE 1 SEQ. ID NO. Nucleic Acid or Protein 1 4F11 Heavy Chain VariableRegion-nucleic acid 2 4F11 Heavy Chain Variable Region-protein 3 4F11Light (kappa) Chain Variable Region-nucleic acid 4 4F11 Light (kappa)Chain Variable Region-protein 5 4F11 Heavy Chain CDR₁ 6 4F11 Heavy ChainCDR₂ 7 4F11 Heavy Chain CDR₃ 8 4F11 Light (kappa) Chain CDR₁ 9 4F11Light (kappa) Chain CDR₂ 10 4F11 Light (kappa) Chain CDR₃

Mouse monoclonal antibody heavy chain CDR sequences (Kabat, Chothia, andIMGT definitions) for 4F11 are shown in Table 2.

TABLE 2 CDR1 CDR2 CDR3 Kabat SYDMS YISRGGGSTYYPDT HATTAYWYFDV (SEQ IDVKG (SEQ ID  NO: 5) (SEQ ID NO: 6) NO: 7) Chothia GFAFSSY SRGGGSHATTAYWYFDV (SEQ ID (SEQ ID NO: 12) (SEQ ID  NO: 11) NO: 7) IMGTGFAFSSYD ISRGGGST GRHATTAYWYFDV (SEQ ID (SEQ ID NO: 15) (SEQ ID  NO: 14)NO: 16)

Mouse monoclonal antibody kappa light chain CDR sequences (Kabat,Chothia, and IMGT definitions) for 4F11 are shown in Table 3.

TABLE 3 CDR1 CDR2 CDR3 Kabat/ RSSQSLVHTNGNTYLH KVSNRFS SQSTHVPWT Chothia(SEQ ID NO: 8) (SEQ ID  (SEQ ID NO: 10) NO: 9) IMGT QSLVHTNGNTY KVSSQSTHVPWT (SEQ ID NO: 20) (SEQ ID NO: 10)

To create the complete heavy or kappa chain antibody sequences, eachvariable sequence above is combined with its respective constant region.For example, a complete heavy chain comprises a heavy variable sequencefollowed by the murine IgG1 heavy chain constant sequence, and acomplete kappa chain comprises a kappa variable sequence followed by themurine kappa light chain constant sequence.

Nucleic Acid Sequence Encoding the Murine IgG1 Heavy Chain ConstantRegion (SEQ ID NO: 22)

  1 gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 61 tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc121 tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac181 ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc241 acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg301 gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc361 cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg421 gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag481 gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc541 agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc601 aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg661 aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc721 agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg781 aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct841 tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc901 acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac961 tctcctggta aa

Protein Sequence Defining the Murine IgG1 Heavy Chain Constant Region(SEQ ID NO: 23)

  1 akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd 61 lytlsssvtv psstwpsetv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif121 ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv181 selpimhqdw lngkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv241 sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf301 tcsvlheglh nhhtekslsh spgk

Nucleic Acid Sequence Encoding the Murine Kappa Light Chain ConstantRegion (SEQ ID NO: 24)

  1 cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 61 ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag121 tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac181 agcaaagaca gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa241 cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag301 agcttcaaca ggaatgagtg t

Protein Sequence Defining the Murine Kappa Light Chain Constant Region(SEQ ID NO: 25)

 1 radaaptvsi fppsseqlts ggasvvcfln nfypkdinvk wkidgserqn gvlnswtdqd 61skdstysmss tltltkdeye rhnsytceat hktstspivk sfnrnec

The following sequences represent the actual or contemplated full lengthheavy and light chain sequences (i.e., containing both the variable andconstant regions sequences) for the 4F11 antibody described in thisExample. Signal sequences for proper secretion of the antibodies (e.g.,signal sequences at the 5′ end of the DNA sequences or the aminoterminal end of the protein sequences) are not shown in the full lengthheavy and light chain sequences disclosed herein and are not included inthe final secreted protein. Also not shown are stop codons fortermination of translation required at the 3′ end of the DNA sequences.It is within ordinary skill in the art to select a signal sequenceand/or a stop codon for expression of the disclosed full length IgGheavy chain and light chain sequences. It is also contemplated that thevariable region sequences can be ligated to other constant regionsequences to produce active full length IgG heavy and light chains.

Nucleic Acid Sequence Encoding the Full Length Heavy Chain Sequence(Heavy Chain Variable Region and IgG1 Constant Region) of 4F11 (SEQ IDNO: 26)

   1 gaagtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc  61 tcctgtgcag cctctggatt cgctttcagt agctatgaca tgtcttgggt tcgccagact 121 ccggagaaga ggctggagtg ggtcgcatac attagtcgtg gtggtggtag cacctactat 181 ccagacactg tgaagggccg attcaccatc tccagagaca atgccaagaa caccctgtac 241 ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgg aagacatgct 301 actacggcct actggtactt cgatgtctgg ggcgcaggga ccacggtcac cgtctcctca 361 gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 421 tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 481 tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 541 ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc 601 acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 661 gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 721 cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 781 gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 841 gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 901 agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 961 aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg1021 aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc1081 agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg1141 aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct1201 tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc1261 acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac1321 tctcctggta aa

Protein Sequence Defining the Full Length Heavy Chain Sequence (HeavyChain Variable Region and IgG1 Constant Region) of 4F11 (SEQ ID NO: 27)

  1 evqlvesggg lvkpggslkl scaasgfafs sydmswvrqt pekrlewvay isrgggstyy 61 pdtvkgrfti srdnakntly lqmsslksed tamyycgrha ttaywyfdvw gagttvtvss121 akttppsvyp lapgsaaqtn smvtlgclvk gyfpepvtvt wnsgslssgv htfpavlqsd181 lytlsssvtv psstwpsetv tcnvahpass tkvdkkivpr dcgckpcict vpevssvfif241 ppkpkdvlti tltpkvtcvv vdiskddpev qfswfvddve vhtaqtqpre eqfnstfrsv301 selpimhqdw ingkefkcrv nsaafpapie ktisktkgrp kapqvytipp pkeqmakdkv361 sltcmitdff peditvewqw ngqpaenykn tqpimdtdgs yfvysklnvq ksnweagntf421 tcsvlheglh nhhtekslsh spgk

Nucleic Acid Sequence Encoding the Full Length Light Chain Sequence(Kappa Chain Variable Region and Constant Region) of 4F11 (SEQ ID NO:28)

  1 gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 61 atctcttgca gatctagtca gagccttgta cacactaatg gcaacaccta tttacattgg121 tacctgcaga agccaggcca gtctccaaaa ctcctgatct acaaagtttc caaccgattt181 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc241 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg301 tggacgttcg gtggaggcac caagctggaa atcaaacggg ctgatgctgc accaactgta361 tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc421 ttgaacaact tctaccccaa agacatcaat gtcaagtgga agattgatgg cagtgaacga481 caaaatggcg tcctgaacag ttggactgat caggacagca aagacagcac ctacagcatg541 agcagcaccc tcacgttgac caaggacgag tatgaacgac ataacagcta tacctgtgag601 gccactcaca agacatcaac ttcacccatt gtcaagagct tcaacaggaa tgagtgt

Protein Sequence Defining the Full Length Light Chain Sequence (KappaChain Variable Region and Constant Region) of 4F11 (SEQ ID NO: 29)

  1 dvvmtqtpls lpvslgdqas iscrssqslv htngntylhw ylqkpgqspk lliykvsnrf 61 sgvpdrfsgs gsgtdftlki srveaedlgv yfcsqsthvp wtfgggtkle ikradaaptv121 sifppsseql tsggasvvcf lnnfypkdin vkwkidgser qngvlnswtd qdskdstysm181 sstltltkde yerhnsytce athktstspi vksfnrnec

Table 4 is a concordance chart showing the correspondence between thefull length sequences of the antibodies discussed in this Example withthose presented in the Sequence Listing.

TABLE 4 SEQ ID NO. Nucleic Acid or Protein 26 4F11 Heavy Variable + IgG1Constant-nucleic acid 27 4F11 Heavy Variable + IgG1 Constant-protein 284F11 Kappa Variable + Constant-nucleic acid 29 4F11 Kappa Variable +Constant-protein

Example 3: Binding Affinities

The binding affinity and kinetics of binding of antibody 4F11 torecombinant human Notch3 extracellular domain (containing EGF likedomains 1-11) Fc fusion protein (rhNotch3-Fc (R&D Systems, Inc.,Minneapolis, Minn.)) were measured by surface plasmon resonance using aBiacore® T100 instrument (GE Healthcare, Piscataway, N.J.).

Rabbit anti-mouse IgGs (GE Healthcare) were immobilized oncarboxymethylated dextran CM4 sensor chips (GE Healthcare) by aminecoupling, according to a standard protocol. Analyses were performed at25° C. and 37° C. using PBS containing 0.05% surfactant P20 as runningbuffer. The antibody was captured in individual flow cells at a flowrate of 10 μl/minute. Injection time was varied for each antibody toyield an Rmax between 30 and 60 resonance units (RU). Buffer orrhNotch3-Fc diluted in running buffer was injected sequentially over areference surface (no antibody captured) and the active surface(antibody to be tested) for 240 seconds at 60 μl/minute. Thedissociation phase was monitored for up to 1500 seconds. The surface wasthen regenerated with two 60-second injections of 10 mM Glycine-HCl, pH2.25, at a flow rate of 30 μl/minute. The rhNotch3-Fc concentrationrange tested was 100 nM to 3.125 nM (2 fold dilution).

Kinetic parameters were determined using the kinetic function of theBIAevaluation software (GE Healthcare) with double referencesubtraction. Kinetic parameters for the antibody, k_(a) (associationrate constant), k_(d) (dissociation rate constant) and K_(D)(equilibrium dissociation constant) were determined. Kinetic values ofthe monoclonal antibody 4F11 on rhNotch3-Fc at 25° C. and 37° C. aresummarized in Table 5.

TABLE 5 Antibody Binding to rhNotch3-Fc Temperature k_(a) (1/Ms) k_(d)(1/s) K_(D) (M) n 25° C. 7.0E+04 4.8E−05 8.23E−10 3 37° C. 7.5E+045.7E−05  7.8E−10 3

The data in Table 5 demonstrate that the antibody 4F11 binds rhNotch3-Fcwith a K_(D) of about 1 nM, 900 pM, 850 pM, 800 pM, 750 pM, 700 pM, 650pM, 600 pM, 500 pM, 400 pM, 300 pM, 250 pM, or 200 pM or less.

Binding to cell surface human Notch3 by the antibody 4F11 was measuredat 4° C., using Fluorescence Activated Cell Sorting (FACS). CHO N3(Flp-In-CHO cells (Invitrogen) stably transfected with human Notch3),HCC2429 cells, and RL-952 cells expressing human Notch3 were washed oncewith PBS containing calcium chloride and magnesium chloride (Invitrogen)and harvested using cell dissociation buffer (Invitrogen). Cells werewashed a second time with PBS and resuspended in FACS buffer (PBS with0.5% BSA (Sigma-Aldrich)) for a final cell concentration of 250,000cells per well into a 96-well v-bottom plate. Purified antibodies werediluted in FACS buffer over a concentration range of 100 nM to 0.1 nM.Cells were then incubated at 4° C. with 100 n1 of antibody for one hour,washed with FACS buffer twice, and resuspended in 100 n1 of goat-antimouse PE-conjugated antibody (Jackson Immuno Research). Cells wereincubated at 4° C. for 30 minutes in the dark, washed once with FACSbuffer, and then analyzed using a Beckman Coulter Cytomics FC 500instrument. The geometric mean of the florescent intensity was thencalculated for each antibody concentration. These values were thenentered into Prism software (GraphPad, La Jolla, Calif.) and used togenerate a binding curve by plotting geometric mean versus antibodyconcentration. From the binding curve, the following equation was usedto calculate the K_(D) and K_(D) range of 4F11 binding to human Notch3on the cell surface of the three cell lines.Y=B _(max) *X/(K _(D) +X)  Equation: One site binding (hyperbola)*describes the binding of a ligand to a receptor that follows the law ofmass action. B_(max) is the maximal binding, and K_(D) is theconcentration of ligand required to reach half-maximal binding.

Results are summarized in Table 6.

TABLE 6 K_(D) (nM) K_(D) Range (nM) CH0 N3 0.36 0.21 to 0.51 HCC24292.58 1.21 to 3.95 RL-952 0.9633 0.7168 to 1.210 

The results in Table 6 demonstrate that the antibody 4F11 binds cellsurface Notch3 with a K_(D) of about 1 nM, 750 pM, 650 pM, 600 pM, 500pM, 400 pM, 300 pM, 250 pM, or 200 pM or less.

Example 4: Binding Specificity

Antibody 4F11 was tested for binding to human Notch1, human Notch2, orhuman Notch3 proteins. Binding measurements were made by biolayerinterferometry (BLI), using a FortéBio Octet® QK instrument (FortéBio,Menlo Park, Calif.). Anti-human-Fc sensors were soaked in PBS containing1 mg/ml BSA for five minutes prior to binding of antibodies. Then thefollowing proteins (400 nM, in PBS containing 1 mg/ml BSA) were allowedto bind to the sensors: rhNotch1-Fc (R&D Systems, Minneapolis, Minn.;Cat. No. 3647-TK-050), rhNotch2-Fc (R&D Cat. No. 3735-NT-050),rhNotch3-Fc (R&D Cat. No. 1559-NT-050), or rmNotch3-Fc (R&D Cat. No.1308-NT-050). Notch protein bound sensors were immersed in antibodysolution (50 μg/ml) to allow binding of antibody to the Notch protein.Binding was detected by shifts in the interference pattern. Theseresults demonstrated that the antibodies bind specifically to humanNotch3 protein, but do not bind to human Notch2 or human Notch1 protein(FIG. 4).

Stable cells lines expressing Notch receptors were produced bytransfecting FlpIn™ CHO or FlpIn™ 293 cells (Life Technologies, GrandIsland, N.Y.) with full length human Notch1, Notch2, Notch3, or Notch4cDNAs cloned into the pcDNA5FRT vector using Lipofectamine 2000 (LifeTechnologies) according to the manufacturer's protocol. Twenty-fourhours after transfection, CHO cells were split into F12 media containing10% FBS, 2 mM L-Glutamine and 700 μg/ml hygromycin B (Sigma-Aldrich, St.Louis, Mo.) to select for transfected cells. 293 cells were split intoDMEM media containing 10% FBS, 2 mM L-glutamine and 200 μg/ml hygromycinB. Expression of Notch receptors was confirmed by FACS analysis usinganti-human Notch1 PE (BioLegend, San Diego, Calif.), anti-Notch2 PE(eBioscience, San Diego, Calif.), or anti-human Notch3 PE (BioLegend,San Diego, Calif.).

To determine specificity of binding to cell surface Notch proteins,antibody 4F11 was tested for binding to human Notch1, human Notch2,human Notch3 and human Notch4 expressed on the surface of CHO cellsusing electrochemiluminescence (Meso Scale Discovery). A CHO linelacking any human Notch protein was also produced for use as a negativecontrol. Cells were grown under standard conditions (37° C., F12+10%FBS). For binding studies, cells were washed in PBS containing calciumand magnesium, and removed from the plate by treatment with CellDissociation Buffer (Life Technologies) for ten minutes at 37° C.

Cells were seeded at a density of 30,000 cells per well, in hybridomamedia, instandard 96-well binding plates (Meso Scale Discovery, Cat. No.L15XA-6). Cells were incubated for one hour at 37° C. Antibodies orcontrol IgG were added at 5 μg/ml, in 50 μl hybridoma media, andincubated for one hour at 37° C. The plates were washed twice with PBScontaining 3% BSA. Binding of the antibodies to cell surface wasdetected using 2 μg/ml of MSD anti-mouse IgG secondary antibody (MesoScale Discovery, Cat. No. R32AC-1) for one hour at 4° C. Plates werewashed twice with PBS containing 3% BSA, and 150 μl of read buffer (MesoScale Discovery Cat. No. R92TC-1) was added. The plates were analyzed ona Sector Imager 2400 instrument (Meso Scale Discovery). This analysisshowed that antibody 4F11 binds to human Notch3 displayed on thesurfaces of cells, but does not bind to human Notch1, Notch2, or Notch4displayed on the surfaces of cells. The 4F11 antibody also does not bindCHO-EV (empty vector) cells that express endogenous hamster Notchproteins. These results indicated that antibody 4F11 binds specificallyto human Notch3 protein displayed on a cell surface in vitro.

Example 5: Inhibition of Notch3-Ligand Binding

The 4F11 antibody was tested for its ability to inhibit the binding ofrhNotch3 binding to human Jag1, Jag2, DLL1 and DLL4. Bindingmeasurements were made by bio-layer interferometry (BLI), using aFortéBio Octet® QK instrument (ForteBio, Menlo Park, Calif.). Theligands tested were rhJag1-Fc (R&D Cat. No. 1277-JG-050), rhJag2-Fc (R&DCat. No. 1726-JG-050), rhDLL1-Fc (R&D Cat. No. 5026-DL-050), and Histagged rhDLL4 (R&D Cat. No. 1506-D4-050).

To determine the degree of inhibition of Notch3-ligand binding byantibody 4F11, the Octet sensors were loaded with recombinant humanNotch3, and the antibody was allowed to bind, as described in Example 4.In positive control samples, a commercially available Notch3 polyclonalantibody capable of blocking ligand binding to recombinant human Notch3(Notch3 Specific Control) was used instead of the 4F11 antibody. Thensensors were immersed in 500 ug/ml human IgG, to block non-specificbinding. Ligands were prepared at a concentration of 400 nM in PBScontaining 3% BSA, and were allowed to bind. The on-rate and off-ratefor ligand binding were detected using the Octet QK instrument andsoftware. The 4F11 antibody blocked binding of all four ligands torhNotch3-Fc (FIG. 5A-5D).

Example 6: Inhibition of Ligand-Induced Notch3 ICD Cleavage

Activation of Notch receptors results in cleavage of the Notchintracellular domain (NICD), which can be detected by Western blot. Theeffect of antibody 4F11 on the activation of Notch3 ICD cleavage wastested.

To create soluble Notch ligands, PCR was used to amplify sequencescorresponding to the extracellular domains of human Jag1 or human Jag2cDNA and fuse them in-frame to the coding sequence of human IgG Fc. Thisconstruct was then subcloned into the pEE14.4 expression vector (Lonza),transfected into CHOK1SV cells, and selected to produce stable celllines that secrete hJag1-hFc or hJag2-hFc fusion protein. 96-wellImmunosorp ELISA plates (Nalgene Nunc, Rochester, N.Y.) were coated with5 μg/ml anti-human Fc (Jackson ImmunoResearch, West Grove, Pa.)overnight at 4° C. After washing wells with PBS/0.5% BSA, 5 μg/ml ofsoluble hJag1-hFc fusion protein was added and allowed to bind at roomtemperature for two hours. Unbound protein was removed by washing withPBS/0.5% BSA. FlpIn™ 293 cells engineered to express hNotch3 (asdescribed in Example 4) were plated on Jag1-hFc ligand or hFc in thepresence of 10 μg/ml 4F11 or mIgG control antibody. Cells were lysed 24hours later in RIPA buffer (Boston BioProducts, Ashland, Mass.)containing protease inhibitors. Induction of cleaved NICD was detectedby probing the blot with a Notch3 antibody against the C-terminus (CellSignaling, Danvers, Mass.) that detects both full length protein and thecleaved ICD. Ligand-induced activation and Notch3 ICD cleavage wasinhibited by antibody 4F11.

Example 7: Inhibition of Notch3-Dependent Transcription

Reporter cell lines dependent upon Notch3 were produced by lentiviralintroduction of a RBP-Jκ-dependent luciferase reporter gene(SABiosciences, Frederick, Md.) into 293-Flpin Notch3 cells, RL95-2endometrial cancer cells, HCC1143 breast cancer cells, and MDA-MB-468breast cancer cells. To activate Notch3-dependent signaling andtranscription, cells were plated on ligand-coated wells prepared asdescribed in Example 6. Cells were pre-incubated with a 3-fold dilutionseries of Notch3 antibody concentrations ranging from 0-300 μg/ml, forone hour at 37° C., before seeding 100 μl of the suspension into 96-wellplates coated with ligand or hFc. Cells were incubated in ligand-coatedor human-Fc-coated wells for four or twenty-four hours at 37° C., in 5%CO₂. Next, 100 μl of Promega Bright Glo™ (Promega, Madison, Wis.) wasadded to each well. The reaction was allowed to proceed for five minutesin the dark, and then the entire 200 μl volume was transferred to whitewalled plates and read using a luminometer. Polyclonal antibodiesagainst Notch1 (AF1057, R&D Systems), Notch2 (AF1190, R&D Systems) orNotch3 (AF1559, R&D Systems) were used as controls to confirm thatligand-stimulated reporter activity in each cell line was specificallydependent upon the introduced Notch receptor. As shown in FIG. 6A,Notch3 antibody 4F11 inhibited Notch3-dependent transcription stimulatedby the ligand Jag2. Activation of Notch3-dependent transcription by eachof the ligands Jag1, Jag2, DLL1, and DLL4 was also inhibited by theNotch3 antibody 4F11 (FIG. 6B and Table 7).

TABLE 7 Ligand Jag1 Jag2 DLL1 DLL4 EC₅₀ 2.6 nM 0.4 nM 6.0 nM 4.7 nMMaximum 92% 99% 100% 73% Inhibition

Example 8: Humanization of Anti-Notch3 Antibodies

This Example describes the humanization and chimerization of theanti-human Notch3 antibody 4F11, and the characterization of theresulting humanized antibodies. The humanized anti-Notch3 antibodieswere designed, affinity matured by targeted CDR mutagenesis, andoptimized using methods known in the art. The amino acid sequences wereconverted to codon-optimized DNA sequences and synthesized to include(in the following order): 5′ HindIII restriction site, Kozak consensussequence, amino terminal signal sequence, humanized variable region,human IgG1 or Kappa constant region, stop codon, and a 3′ EcoRIrestriction site.

Chimeric (murine variable region and human constant region) 4F11 heavy(human IgG1) and light (human Kappa) chains were also constructed. Togenerate chimeric antibodies, the murine variable regions were fused tothe human constant region, and codon-optimized DNA sequences weresynthesized, including (in the following order): 5′ HindIII restrictionsite, Kozak consensus sequence, amino terminal signal sequence, mousevariable region, human IgG1 or Kappa constant region, stop codon, and 3′EcoRI restriction site.

The humanized and chimeric heavy chains were subcloned into pEE6.4(Lonza, Basel, Switzerland) via HindIII and EcoRI sites using In-Fusion™PCR cloning (Clontech, Mountain View, Calif.). The humanized andchimeric Kappa light chains were subcloned into pEE14.4 (Lonza) viaHindIII and EcoRI sites using In-Fusion™ PCR cloning.

Humanized antibody chains or chimeric antibody chains were transientlytransfected into 293T cells to produce antibody. Antibody was eitherpurified or used in cell culture media supernatant for subsequent invitro analysis. Binding of the chimeric and humanized antibodies toNotch3 was measured as described below. The results are summarized inTables 14-16.

Exemplary combinations of the chimeric or humanized 4F11 immunoglobulinheavy chain and immunoglobulin light chain variable regions is set forthbelow in Table 8.

TABLE 8 Antibody Name Heavy Chain Variable Region Light Chain VariableRegion Hu4F11-1 Ch4F11 Chimeric Heavy Ch4F11 Chimeric Kappa (SEQ ID NO:2) (SEQ ID NO: 4) Hu4F11-10 Sh4F11 Hv3-23 Heavy Ch4F11 Chimeric Kappa(SEQ ID NO: 34) (SEQ ID NO: 4) Hu4F11-18 Ch4F11 Chimeric Heavy Hu4F11Kv2D-29 Kappa (SEQ ID NO: 2) (SEQ ID NO: 44) Hu4F11-32 Sh4F11 Hv3-23Heavy Hu4F11 Kv2D-29 Kappa (SEQ ID NO: 34) (SEQ ID NO: 44) Hu4F11-69Sh4F11 Hv3-23 A28T S31N T62S Heavy Hu4F11 Kv2D-29 N28H Kappa (SEQ ID NO:40) (SEQ ID NO: 46) Hu4F11-70 Sh4F11 Hv3-23 A28T S31H T62S Heavy Hu4F11Kv2D-29 N28H Kappa (SEQ ID NO: 36) (SEQ ID NO: 46) Hu4F11-71 Sh4F11Hv3-23 A28T T62S Heavy Hu4F11 Kv2D-29 N28H Kappa (SEQ ID NO: 42) (SEQ IDNO: 46) Hu4F11-72 Sh4F11 Hv3-23 S31H T62S Heavy Hu4F11 Kv2D-29 N28HKappa (SEQ ID NO: 38) (SEQ ID NO: 46) Hu4F11-73 Sh4F11 Hv3-23 A28T S31NT62S Heavy Hu4F11 Kv2D-29 N28Y Kappa (SEQ ID NO: 40) (SEQ ID NO: 50)Hu4F11-74 Sh4F11 Hv3-23 A28T S31H T62S Heavy Hu4F11 Kv2D-29 N28Y Kappa(SEQ ID NO: 36) (SEQ ID NO: 50) Hu4F11-75 Sh4F11 Hv3-23 A28T T62S HeavyHu4F11 Kv2D-29 N28Y Kappa (SEQ ID NO: 42) (SEQ ID NO: 50) Hu4F11-76Sh4F11 Hv3-23 S31H T62S Heavy Hu4F11 Kv2D-29 N28Y Kappa (SEQ ID NO: 38)(SEQ ID NO: 50) Hu4F11-77 Sh4F11 Hv3-23 A28T S31N T62S Heavy Hu4F11Kv2D-29 N28Q Kappa (SEQ ID NO: 40) (SEQ ID NO: 48) Hu4F11-78 Sh4F11Hv3-23 A28T S31H T62S Heavy Hu4F11 Kv2D-29 N28Q Kappa (SEQ ID NO: 36)(SEQ ID NO: 48) Hu4F11-79 Sh4F11 Hv3-23 A28T T62S Heavy Hu4F11 Kv2D-29N28Q Kappa (SEQ ID NO: 42) (SEQ ID NO: 48) Hu4F11-80 Sh4F11 Hv3-23 S31HT62S Heavy Hu4F11 Kv2D-29 N28Q Kappa (SEQ ID NO: 38) (SEQ ID NO: 48)

The nucleic acid sequences and the encoded protein sequences definingvariable regions of the chimeric and humanized 4F11 antibodies aresummarized below (amino terminal signal peptide sequences are notshown). CDR sequences (Kabat definition) are shown in bold and areunderlined in the amino acid sequences.

Nucleic Acid Sequence Encoding the Ch4F11 Chimeric Heavy Chain VariableRegion (SEQ ID NO:31)

  1 gaggtacagc ttgtcgagtc gggaggagga ttggtaaaac cgggtgggtc actcaaattg   61 tcgtgtgcgg cgtcgggatt tgcgttttcg tcgtatgata tgtcgtgggt gcgccagacg121 ccggaaaaac gattggaatg ggtcgcgtat atctcccgag ggggaggttc gacatactat181 cccgacacgg tcaaagggcg cttcacgatt tcacgggaca atgcgaaaaa cacgctttat241 cttcaaatgt cgtcgttgaa atcggaagat accgcgatgt attactgcgg gaggcatgcg301 acgacggcgt attggtattt cgatgtgtgg ggagccggaa cgacggtgac ggtgtcgtcg

Protein Sequence Defining the Ch4F11 Chimeric Heavy Chain VariableRegion (SEQ ID NO:2)

 1 evqlvesggg lvkpggslkl scaasgfafs  sydms wvrqt pekrlewva y  isrgggstyy 61 pdtvkg rfti srdnakntly lqmsslksed tamyycgr ha   ttaywyfdvw gagttvtvss

Nucleic Acid Sequence Encoding the Sh4F11 Hv3-23 Heavy Chain VariableRegion (SEQ ID NO:33)

  1 gaagtacagt tgttggagtc aggaggaggg ttggtccagc cgggtggatc gttgcggctt 61 tcgtgtgcgg cgtcgggatt cgcgttttca tcgtatgaca tgtcgtgggt gaggcaggca121 ccggggaaag ggcttgaatg ggtatcgtac atttcgagag ggggaggatc gacgtattac181 ccggatacgg tgaaaggaag gtttacgatc tcgcgcgaca attcaaagaa tacgctttat241 cttcagatga actcgctccg agcggaagat acggcggtat actattgcgg tcgccatgcg301 acgacggcgt attggtattt cgatgtgtgg ggacaaggga cgatggtcac ggtgtcgtcg

Protein Sequence Defining the Sh4F11 Hv3-23 Heavy Chain Variable Region(SEQ ID NO:34)

 1 evqllesggg lvqpggslrl scaasgfafs  sydms wvrqa pgkglewvs y  isrgggstyy 61 pdtvkg rfti srdnskntly lqmnslraed tavyycgr ha   ttaywyfdvw gqgtmvtvss

Nucleic Acid Sequence Encoding the Sh4F11 Hv3-23 A28T S31H T62S HeavyChain Variable Region (SEQ ID NO:35)

  1 gaagtacagt tgttggagtc aggaggaggg ttggtccagc cgggtggatc gttgcggctt 61 tcgtgtgcgg cgtcgggatt caccttttca cactatgaca tgtcgtgggt gaggcaggca121 ccggggaaag ggcttgaatg ggtatcgtac atttcgagag ggggaggatc gacgtattac181 ccggattccg tgaaaggaag gtttacgatc tcgcgcgaca attcaaagaa tacgctttat241 cttcagatga actcgctccg agcggaagat acggcggtat actattgcgg tcgccatgcg301 acgacggcgt attggtattt cgatgtgtgg ggacaaggga cgatggtcac ggtgtcgtcg

Protein Sequence Defining the Sh4F11 Hv3-23 A28T S31H T62S Heavy ChainVariable Region (SEQ ID NO:36)

 1 evqllesggg lvqpggslrl scaasgftfs  hydms wvrqa pgkglewvs y  isrgggstyy 61 pdsvkg rfti srdnskntly lqmnslraed tavyycgr ha   ttaywyfdvw gqgtmvtvss

Nucleic Acid Sequence Encoding the Sh4F11 Hv3-23 S31H T62S Heavy ChainVariable Region (SEQ ID NO:37)

1 gaagtacagt tgttggagtc aggaggaggg ttggtccagc cgggtggatc gttgcggctt 61tcgtgtgcgg cgtcgggatt cgcgttttca cactatgaca tgtcgtgggt gaggcaggca 121ccggggaaag ggcttgaatg ggtatcgtac atttcgagag ggggaggatc gacgtattac 181ccggattccg tgaaaggaag gtttacgatc tcgcgcgaca attcaaagaa tacgctttat 241cttcagatga actcgctccg agcggaagat acggcggtat actattgcgg tcgccatgcg 301acgacggcgt attggtattt cgatgtgtgg ggacaaggga cgatggtcac ggtgtcgtcg

Protein Sequence Defining the Sh4F11 Hv3-23 S31H T62S Heavy ChainVariable Region (SEQ ID NO:38)

1 evqllesggg lvqpggslrl scaasgfafs  hydms wvrqa pgkglewvs y   isrgggstyy61 pdsvkg rfti srdnskntly lqmnslraed tavyycgr ha ttaywyfdv w gqgtmvtvss

Nucleic Acid Sequence Encoding the Sh4F11 Hv3-23 A28T S31N T62S HeavyChain Variable Region (SEQ ID NO:39)

1 gaagtacagt tgttggagtc aggaggaggg ttggtccagc cgggtggatc gttgcggctt 61tcgtgtgcgg cgtcgggatt caccttttca aactatgaca tgtcgtgggt gaggcaggca 121ccggggaaag ggcttgaatg ggtatcgtac atttcgagag ggggaggatc gacgtattac 181ccggattccg tgaaaggaag gtttacgatc tcgcgcgaca attcaaagaa tacgctttat 241cttcagatga actcgctccg agcggaagat acggcggtat actattgcgg tcgccatgcg 301acgacggcgt attggtattt cgatgtgtgg ggacaaggga cgatggtcac ggtgtcgtcg

Protein Sequence Defining the Sh4F11 Hv3-23 A28T S31N T62S Heavy ChainVariable Region (SEQ ID NO:40)

1 evqllesggg lvqpggslrl scaasgftfs  nydms wvrqa pgkglewvs y   isrgggstyy61 pdsvkg rfti srdnskntly lqmnslraed tavyycgr ha ttaywyfdv w gqgtmvtvss

Nucleic Acid Sequence Encoding the Sh4F11 Hv3-23 A28T T62S Heavy ChainVariable Region (SEQ ID NO:41)

1 gaagtacagt tgttggagtc aggaggaggg ttggtccagc cgggtggatc gttgcggctt 61tcgtgtgcgg cgtcgggatt caccttttca tcgtatgaca tgtcgtgggt gaggcaggca 121ccggggaaag ggcttgaatg ggtatcgtac atttcgagag ggggaggatc gacgtattac 181ccggattccg tgaaaggaag gtttacgatc tcgcgcgaca attcaaagaa tacgctttat 241cttcagatga actcgctccg agcggaagat acggcggtat actattgcgg tcgccatgcg 301acgacggcgt attggtattt cgatgtgtgg ggacaaggga cgatggtcac ggtgtcgtcg

Protein Sequence Defining the Sh4F11 Hv3-23 A28T T62S Heavy ChainVariable Region (SEQ ID NO:42)

1 evqllesggg lvqpggslrl scaasgftfs  sydms wvrqa pgkglewvs y   isrgggstyy61 pdsvkg rfti srdnskntly lqmnslraed tavyycgr ha ttaywyfdv w gqgtmvtvss

Nucleic Acid Sequence Encoding the Ch4F11 Chimeric Kappa Chain VariableRegion (SEQ ID NO:32)

1 gacgtggtaa tgacgcagac gccgttgtcc cttcctgtct cgctcggaga tcaggcgtcg 61atctcgtgta gaagctcgca gtcactcgtc cataccaacg ggaatacata tcttcactgg 121tatttgcaaa agcccggaca gtcaccgaag ctcttgatct acaaagtatc caatcggttt 181tcgggggtgc ccgaccgatt ctcgggatcg ggttcgggga cggattttac gttgaagatt 241tcgcgggtgg aagcggagga tctcggtgtc tacttttgtt cgcagtcaac gcatgtcccg 301tggacgttcg gaggcgggac aaaacttgag atcaag

Protein Sequence Defining the Ch4F11 Chimeric Kappa Chain VariableRegion (SEQ ID NO:4)

1 dvvmtgtpls lpvslgdqas isc rssqslv   htngntylh w ylqkpgqspk lliy kvsnrf61 s gvpdrfsgs gsgtdftlki srveaedlgv yfc sqsthvp wt fgggtkle ik

Nucleic Acid Sequence Encoding the Hu4F11 Kv2D-29 Kappa Chain VariableRegion (SEQ ID NO:43)

1 gatgtagtca tgacccaaac gccgctttcg ttgtcggtga cgcccggaca gcccgcgtca 61atctcgtgtc ggtcatcgca gtcgttggta cacacaaacg gtaatacgta tctccattgg 121tatctccaga agcccggcca gtcgccgcag ctcttgatct acaaagtgag caatcgcttt 181tcgggggtgc cggatcggtt ctcgggatcg gggtcaggaa cggacttcac gcttaagatt 241tcgagggtcg aggcggagga tgtcggagtc tacttttgtt cgcagtccac acatgtcccc 301tggacgtttg ggcaggggac gaaggtggaa atcaag

Protein Sequence Defining the Hu4F11 Kv2D-29 Kappa Chain Variable Region(SEQ ID NO:44)

1 dvvmtqtpls lsvtpgqpas isc rss q slv   htngntylh w ylqkpgqspq lliykvsnrf 61 s gvpdrfsgs gsgtdftlki srveaedvgv yfc sqsthvp wt fgqgtkve ik

Nucleic Acid Sequence Encoding the Hu4F11 Kv2D-29 N28H Kappa ChainVariable Region (SEQ ID NO:45)

1 gatgtagtca tgacccaaac gccgctttcg ttgtcggtga cgcccggaca gcccgcgtca 61atctcgtgtc ggtcatcgca gtcgttggta cacacacacg gtaatacgta tctccattgg 121tatctccaga agcccggcca gtcgccgcag ctcttgatct acaaagtgag caatcgcttt 181tcgggggtgc cggatcggtt ctcgggatcg gggtcaggaa cggacttcac gcttaagatt 241tcgagggtcg aggcggagga tgtcggagtc tacttttgtt cgcagtccac acatgtcccc 301tggacgtttg ggcaggggac gaaggtggaa atcaag

Protein Sequence Defining the Hu4F11 Kv2D-29 N28H Kappa Chain VariableRegion (SEQ ID NO:46)

1 dvvmtqtpls lsvtpgqpas isc rss q slv   hthgntylh w ylqkpgqspq lliykvsnrf 61 s gvpdrfsgs gsgtdftlki srveaedvgv yfc s q sthvp wt fgqgtkve ik

Nucleic Acid Sequence Encoding the Hu4F11 Kv2D-29 N28Q Kappa ChainVariable Region (SEQ ID NO:47)

1 gatgtagtca tgacccaaac gccgctttcg ttgtcggtga cgcccggaca gcccgcgtca 61atctcgtgtc ggtcatcgca gtcgttggta cacacacaag gtaatacgta tctccattgg 121tatctccaga agcccggcca gtcgccgcag ctcttgatct acaaagtgag caatcgcttt 181tcgggggtgc cggatcggtt ctcgggatcg gggtcaggaa cggacttcac gcttaagatt 241tcgagggtcg aggcggagga tgtcggagtc tacttttgtt cgcagtccac acatgtcccc 301tggacgtttg ggcaggggac gaaggtggaa atcaag

Protein Sequence Defining the Hu4F11 Kv2D-29 N28Q Kappa Chain VariableRegion (SEQ ID NO:48)

1 dvvmtqtpls lsvtpgqpas isc rssqslv   htqgntylh w ylqkpgqspq lliy kvsnrf61 s gvpdrfsgs gsgtdftlki srveaedvgv yfc sqsthvp wt fgqgtkve ik

Nucleic Acid Sequence Encoding the Hu4F11 Kv2D-29 N28Y Kappa ChainVariable Region (SEQ ID NO:49)

1 gatgtagtca tgacccaaac gccgctttcg ttgtcggtga cgcccggaca gcccgcgtca 61atctcgtgtc ggtcatcgca gtcgttggta cacacatacg gtaatacgta tctccattgg 121tatctccaga agcccggcca gtcgccgcag ctcttgatct acaaagtgag caatcgcttt 181tcgggggtgc cggatcggtt ctcgggatcg gggtcaggaa cggacttcac gcttaagatt 241tcgagggtcg aggcggagga tgtcggagtc tacttttgtt cgcagtccac acatgtcccc 301tggacgtttg ggcaggggac gaaggtggaa atcaag

Protein Sequence Defining the Hu4F11 Kv2D-29 N28Y Kappa Chain VariableRegion (SEQ ID NO:50)

1 dvvmtqtpls lsvtpgqpas isc rssqslv   htygntylh w ylqkpgqspq lliy kvsnrf61 s gvpdrfsgs gsgtdftlki srveaedvgv yfc sqsthv p  wt fgqgtkve ik

The amino acid sequences defining the immunoglobulin heavy chainvariable regions for the antibodies produced in Example 8 are aligned inFIG. 7. Amino terminal signal peptide sequences (for properexpression/secretion) are not shown. CDR₁, CDR₂, and CDR₃ (Kabatdefinition) are identified by boxes. FIG. 8 show an alignment of theseparate CDR₁, CDR₂, and CDR₃ sequences for each of the variable regionsequences shown in FIG. 7.

The amino acid sequences defining the immunoglobulin light chainvariable regions for the antibodies in Example 8 are aligned in FIG. 9Amino terminal signal peptide sequences (for properexpression/secretion) are not shown. CDR₁, CDR₂ and CDR₃ are identifiedby boxes. FIG. 10 shows an alignment of the separate CDR₁, CDR₂, andCDR₃ sequences for each of the variable region sequences shown in FIG.9.

Table 9 is a concordance chart showing the SEQ ID NO. of each sequencediscussed in this Example.

TABLE 9 SEQ. ID NO. Nucleic Acid or Protein 31 Ch4F11 Chimeric HeavyChain Variable Region-nucleic acid 2 Ch4F11 Chimeric Heavy ChainVariable Region-protein 5 Ch4F11 Chimeric Heavy Chain CDR₁ 6 Ch4F11Chimeric Heavy Chain CDR₂ 7 Ch4F11 Chimeric Heavy Chain CDR₃ 33 Sh4F11Hv3-23 Heavy Chain Variable Region-nucleic acid 34 Sh4F11 Hv3-23 HeavyChain Variable Region-protein 5 Sh4F11 Hv3-23 Heavy Chain CDR₁ 6 Sh4F11Hv3-23 Heavy Chain CDR₂ 7 Sh4F11 Hv3-23 Heavy Chain CDR₃ 35 Sh4F11Hv3-23 A28T S31H T62S Heavy Chain Variable Region-nucleic acid 36 Sh4F11Hv3-23 A28T S31H T62S Heavy Chain Variable Region-protein 51 Sh4F11Hv3-23 A28T S31H T62S Heavy Chain CDR₁ 53 Sh4F11 Hv3-23 A28T S31H T62SHeavy Chain CDR₂ 7 Sh4F11 Hv3-23 A28T S31H T62S Heavy Chain CDR₃ 37Sh4F11 Hv3-23 S31H T62S Heavy Chain Variable Region-nucleic acid 38Sh4F11 Hv3-23 S31H T62S Heavy Chain Variable Region-protein 51 Sh4F11Hv3-23 S31H T62S Heavy Chain CDR₁ 53 Sh4F11 Hv3-23 S31H T62S Heavy ChainCDR₂ 7 Sh4F11 Hv3-23 S31H T62S Heavy Chain CDR₃ 39 Sh4F11 Hv3-23 A28TS31N T62S Heavy Chain Variable Region-nucleic acid 40 Sh4F11 Hv3-23 A28TS31N T62S Heavy Chain Variable Region-protein 52 Sh4F11 Hv3-23 A28T S31NT62S Heavy Chain CDR₁ 53 Sh4F11 Hv3-23 A28T S31N T62S Heavy Chain CDR₂ 7Sh4F11 Hv3-23 A28T S31N T62S Heavy Chain CDR₃ 41 Sh4F11 Hv3-23 A28T T62SHeavy Chain Variable Region-nucleic acid 42 Sh4F11 Hv3-23 A28T T62SHeavy Chain Variable Region-protein 5 Sh4F11 Hv3-23 A28T T62S HeavyChain CDR₁ 53 Sh4F11 Hv3-23 A28T T62S Heavy Chain CDR₂ 7 Sh4F11 Hv3-23A28T T62S Heavy Chain CDR₃ 32 Ch4F11 Chimeric Light (kappa) ChainVariable Region-nucleic acid 4 Ch4F11 Chimeric Light (kappa) ChainVariable Region-protein 8 Ch4F11 Chimeric Light (kappa) Chain CDR₁ 9Ch4F11 Chimeric Light (kappa) Chain CDR₂ 10 Ch4F11 Chimeric Light(kappa) Chain CDR₃ 43 Hu4F11 Kv2D-29 Light (kappa) Chain VariableRegion-nucleic acid 44 Hu4F11 Kv2D-29 Light (kappa) Chain VariableRegion-protein 8 Hu4F11 Kv2D-29 Light (kappa) Chain CDR₁ 9 Hu4F11Kv2D-29 Light (kappa) Chain CDR₂ 10 Hu4F11 Kv2D-29 Light (kappa) ChainCDR₃ 45 Hu4F11 Kv2D-29 N28H Light (kappa) Chain Variable Region-nucleicacid 46 Hu4F11 Kv2D-29 N28H Light (kappa) Chain Variable Region-protein62 Hu4F11 Kv2D-29 N28H Light (kappa) Chain CDR₁ 9 Hu4F11 Kv2D-29 N28HLight (kappa) Chain CDR₂ 10 Hu4F11 Kv2D-29 N28H Light (kappa) Chain CDR₃47 Hu4F11 Kv2D-29 N28Q Light (kappa) Chain Variable Region-nucleic acid48 Hu4F11 Kv2D-29 N28Q Light (kappa) Chain Variable Region-protein 63Hu4F11 Kv2D-29 N28Q Light (kappa) Chain CDR₁ 9 Hu4F11 Kv2D-29 N28Q Light(kappa) Chain CDR₂ 10 Hu4F11 Kv2D-29 N28Q Light (kappa) Chain CDR₃ 49Hu4F11 Kv2D-29 N28Y Light (kappa) Chain Variable Region-nucleic acid 50Hu4F11 Kv2D-29 N28Y Light (kappa) Chain Variable Region-protein 64Hu4F11 Kv2D-29 N28Y Light (kappa) Chain CDR₁ 9 Hu4F11 Kv2D-29 N28Y Light(kappa) Chain CDR₂ 10 Hu4F11 Kv2D-29 N28Y Light (kappa) Chain CDR₃

Humanized monoclonal antibody heavy chain CDR sequences (Kabat, Chothia,and IMGT definitions) are shown in Table 10.

TABLE 10 Variable Region CDR1 CDR2 CDR3 SEQ ID NO: Kabat Ch4F11 SYDMSYISRGGGSTYYPDTVKG HATTAYWYFDV  2 Chimeric (SEQ ID NO: 5) (SEQ ID NO: 6)(SEQ ID NO: 7) Sh4F11 SYDMS YISRGGGSTYYPDTVKG HATTAYWYFDV 34 HV3-23(SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) Sh4F11 HYDMSYISRGGGSTYYPDSVKG HATTAYWYFDV 36 Hv3-23 A28T (SEQ ID NO: 51)(SEQ ID NO: 53) (SEQ ID NO: 7) S31H T62S Sh4F11 HYDMS YISRGGGSTYYPDSVKGHATTAYWYFDV 38 Hv3-23 S31H (SEQ ID NO: 51) (SEQ ID NO: 53)(SEQ ID NO: 7) T62S Sh4F11 NYDMS YISRGGGSTYYPDSVKG HATTAYWYFDV 40Hv3-23 A28T (SEQ ID NO: 52) (SEQ ID NO: 53) (SEQ ID NO: 7) S31N T62SSh4F11 SYDMS YISRGGGSTYYPDSVKG HATTAYWYFDV 42 Hv3-23 A28T (SEQ ID NO: 5)(SEQ ID NO: 53) (SEQ ID NO: 7) T62S Chothia Ch4F11 GFAFSSY SRGGGSHATTAYWYFDV  2 Chimeric (SEQ ID NO: 11) (SEQ ID NO: 12) (SEQ ID NO: 7)Sh4F11 GFAFSSY SRGGGS HATTAYWYFDV 34 Hv3-23 (SEQ ID NO: 11)(SEQ ID NO: 12) (SEQ ID NO: 7) Sh4F11 GFTFSHY SRGGGS HATTAYWYFDV 36Hv3-23 A28T (SEQ ID NO: 54) (SEQ ID NO: 12) (SEQ ID NO: 7) S31H T62SSh4F11 GFAFSHY SRGGGS HATTAYWYFDV 38 Hv3-23 S31H (SEQ ID NO: 55)(SEQ ID NO: 12) (SEQ ID NO: 7) T62S Sh4F11 GFTFSNY SRGGGS HATTAYWYFDV 40Hv3-23 A28T (SEQ ID NO: 56) (SEQ ID NO: 12) (SEQ ID NO: 7) S31N T62SSh4F11 GFTFSSY SRGGGS HATTAYWYFDV 42 Hv3-23 A28T (SEQ ID NO: 57)(SEQ ID NO: 12) (SEQ ID NO: 7) T62S IMGT Ch4F11 GFAFSSYD ISRGGGSTGRHATTAYWYFDV  2 Chimeric (SEQ ID NO: 14) (SEQ ID NO: 15)(SEQ ID NO: 16) Sh4F11 GFAFSSYD ISRGGGST GRHATTAYWYFDV 34 Hv3-23(SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16) Sh4F11 GFTFSHYD ISRGGGSTGRHATTAYWYFDV 36 Hv3-23 A28T (SEQ ID NO:58) (SEQ ID NO: 15)(SEQ ID NO: 16) S31H T62S Sh4F11 GFAFSHYD ISRGGGST GRHATTAYWYFDV 38Hv3-23 S31H (SEQ ID NO:59) (SEQ ID NO: 15) (SEQ ID NO: 16) T62S Sh4F11GFTFSNYD ISRGGGST GRHATTAYWYFDV 40 Hv3-23 A28T (SEQ ID NO:60)(SEQ ID NO: 15) (SEQ ID NO: 16) S31N T62S Sh4F11 GFTFSSYD ISRGGGSTGRHATTAYWYFDV 42 Hv3-23 A28T (SEQ ID NO:61) (SEQ ID NO: 15)(SEQ ID NO: 16) T62S

Humanized monoclonal antibody Kappa light chain CDR sequences (Kabat,Chothia, and IMGT definitions) are shown in Table 11.

TABEL 11 Variable Region CDR1 CDR2 CDR3 SEQ ID NO: Kabat/Chothia Ch4F11RSSQSLVHTNGNTYLH KVSNRFS SQSTHVPWT  4 Chimeric (SEQ ID NO: 8)(SEQ ID NO: 9) (SEQ ID NO: 10) Hu4F11 RSSQSLVHTNGNTYLH KVSNRFS SQSTHVPWT44 Kv2D-29 (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) Hu4F11RSSQSLVHTHGNTYLH KVSNRFS SQSTHVPWT 46 Kv2D-29 (SEQ ID NO: 62)(SEQ ID NO: 9) (SEQ ID NO: 10) N28H Hu4F11 RSSQSLVHTQGNTYLH KVSNRFSSQSTHVPWT 48 Kv2D-29 (SEQ ID NO: 63) (SEQ ID NO: 9) (SEQ ID NO: 10) N28QHu4F11 RSSQSLVHTYGNTYLH KVSNRFS SQSTHVPWT 50 Kv2D-29 (SEQ ID NO: 64)(SEQ ID NO: 9) (SEQ ID NO: 10) N28Y IMGT Ch4F11 QSLVHTNGNTY KVSSQSTHVPWT  4 Chimeric (SEQ ID NO: 20) (SEQ ID NO: 10) Hu4F11 QSLVHTNGNTYKVS SQSTHVPWT 44 Kv2D-29 (SEQ ID NO: 20) (SEQ ID NO: 10) Hu4F11QSLVHTHGNTY KVS SQSTHVPWT 46 Kv2D-29 (SEQ ID NO: 65) (SEQ ID NO: 10)N28H Hu4F11 QSLVHTQGNTY KVS SQSTHVPWT 48 Kv2D-29 (SEQ ID NO: 66)(SEQ ID NO: 10) N28Q Hu4F11 QSLVHTYGNTY KVS SQSTHVPWT 50 Kv2D-29(SEQ ID NO: 67) (SEQ ID NO: 10) N28Y

To create the complete chimeric and humanized heavy or kappa chainantibody sequences, each variable sequence above is combined with itsrespective human constant region. For example, a complete heavy chaincomprises a heavy variable sequence followed by a human IgG1 heavy chainconstant sequence. A complete kappa chain comprises a kappa variablesequence followed by the human kappa light chain constant sequence.

Nucleic Acid Sequence Encoding the Human IgG1 Heavy Chain ConstantRegion (SEQ ID NO:68)

1 gcctcaacaa aaggaccaag tgtgttccca ctcgccccta gcagcaagag tacatccggg 61ggcactgcag cactcggctg cctcgtcaag gattattttc cagagccagt aaccgtgagc 121tggaacagtg gagcactcac ttctggtgtc catacttttc ctgctgtcct gcaaagctct 181ggcctgtact cactcagctc cgtcgtgacc gtgccatctt catctctggg cactcagacc 241tacatctgta atgtaaacca caagcctagc aatactaagg tcgataagcg ggtggaaccc 301aagagctgcg acaagactca cacttgtccc ccatgccctg cccctgaact tctgggcggt 361cccagcgtct ttttgttccc accaaagcct aaagatactc tgatgataag tagaacaccc 421gaggtgacat gtgttgttgt agacgtttcc cacgaggacc cagaggttaa gttcaactgg 481tacgttgatg gagtcgaagt acataatgct aagaccaagc ctagagagga gcagtataat 541agtacatacc gtgtagtcag tgttctcaca gtgctgcacc aagactggct caacggcaaa 601gaatacaaat gcaaagtgtc caacaaagca ctcccagccc ctatcgagaa gactattagt 661aaggcaaagg ggcagcctcg tgaaccacag gtgtacactc tgccacccag tagagaggaa 721atgacaaaga accaagtctc attgacctgc ctggtgaaag gcttctaccc cagcgacatc 781gccgttgagt gggagagtaa cggtcagcct gagaacaatt acaagacaac ccccccagtg 841ctggatagtg acgggtcttt ctttctgtac agtaagctga ctgtggacaa gtcccgctgg 901cagcagggta acgtcttcag ctgttccgtg atgcacgagg cattgcacaa ccactacacc 961cagaagtcac tgagcctgag cccagggaag

Protein Sequence Defining the Human IgG1 Heavy Chain Constant Region(SEQ ID NO:69)

1 astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss 61glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg 121psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn 181styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree 241mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 301qqgnvfscsv mhealhnhyt qkslslspgk

Nucleic Acid Sequence Encoding the Human Kappa Light Chain ConstantRegion (Version 1) (SEQ ID NO:70)

1 cgcacagttg ctgcccccag cgtgttcatt ttcccaccta gcgatgagca gctgaaaagc 61ggtactgcct ctgtcgtatg cttgctcaac aacttttacc cacgtgaggc taaggtgcag 121tggaaagtgg ataatgcact tcaatctgga aacagtcaag agtccgtgac agaacaggac 181agcaaagact caacttattc actctcttcc accctgactc tgtccaaggc agactatgaa 241aaacacaagg tatacgcctg cgaggttaca caccagggtt tgtctagtcc tgtcaccaag 301tccttcaata ggggcgaatg t

Nucleic Acid Sequence Encoding the Human Kappa Light Chain ConstantRegion (Version 2) (SEQ ID NO:71)

1 cgcacagttg cagcccccag cgtgttcatt ttcccaccta gcgatgagca gctgaaaagc 61ggtactgcct ctgtcgtatg cttgctcaac aacttttacc cacgtgaggc taaggtgcag 121tggaaagtgg ataatgcact tcaatctgga aacagtcaag agtccgtgac agaacaggac 181agcaaagact caacttattc actctcttcc accctgactc tgtccaaggc agactatgaa 241aaacacaagg tatacgcctg cgaggttaca caccagggtt tgtctagtcc tgtcaccaag 301tccttcaata ggggcgaatg t

Protein Sequence Defining the Human Kappa Light Chain Constant Region(nucleic acid versions 1 and 2 encode the same amino acid sequence) (SEQID NO:72)

1 rtvaapsvfi fppsdeqlks gtasvvclln nfypreakvq wkvdnalqsg nsqesvteqd 61skdstyslss tltlskadye khkvyacevt hqglsspvtk sfnrgec

The following sequences represent the actual or contemplated full lengthheavy and light chain sequence (i.e., containing both the variable andconstant regions sequences) for each antibody described in this Example.Signal sequences for proper secretion of the antibodies (e.g., signalsequences at the 5′ end of the DNA sequences or the amino terminal endof the protein sequences) are not shown in the full length heavy andlight chain sequences disclosed herein and are not included in the finalsecreted protein. Also not shown are stop codons for termination oftranslation required at the 3′ end of the DNA sequences. It is withinordinary skill in the art to select a signal sequence and/or a stopcodon for expression of the disclosed full length immunoglobulin heavychain and light chain sequences. It is also contemplated that thevariable region sequences can be ligated to other constant regionsequences to produce active full length immunoglobulin heavy and lightchains.

Nucleic Acid Sequence Encoding the Full Length Ch4F11 Chimeric HeavyChain (Mouse Heavy Chain Variable Region and Human IgG1 Constant Region)(SEQ ID NO:73)

1 gaggtacagc ttgtcgagtc gggaggagga ttggtaaaac cgggtgggtc actcaaattg 61tcgtgtgcgg cgtcgggatt tgcgttttcg tcgtatgata tgtcgtgggt gcgccagacg 121ccggaaaaac gattggaatg ggtcgcgtat atctcccgag ggggaggttc gacatactat 181cccgacacgg tcaaagggcg cttcacgatt tcacgggaca atgcgaaaaa cacgctttat 241cttcaaatgt cgtcgttgaa atcggaagat accgcgatgt attactgcgg gaggcatgcg 301acgacggcgt attggtattt cgatgtgtgg ggagccggaa cgacggtgac ggtgtcgtcg 361gcctcaacaa aaggaccaag tgtgttccca ctcgccccta gcagcaagag tacatccggg 421ggcactgcag cactcggctg cctcgtcaag gattattttc cagagccagt aaccgtgagc 481tggaacagtg gagcactcac ttctggtgtc catacttttc ctgctgtcct gcaaagctct 541ggcctgtact cactcagctc cgtcgtgacc gtgccatctt catctctggg cactcagacc 601tacatctgta atgtaaacca caagcctagc aatactaagg tcgataagcg ggtggaaccc 661aagagctgcg acaagactca cacttgtccc ccatgccctg cccctgaact tctgggcggt 721cccagcgtct ttttgttccc accaaagcct aaagatactc tgatgataag tagaacaccc 781gaggtgacat gtgttgttgt agacgtttcc cacgaggacc cagaggttaa gttcaactgg 841tacgttgatg gagtcgaagt acataatgct aagaccaagc ctagagagga gcagtataat 901agtacatacc gtgtagtcag tgttctcaca gtgctgcacc aagactggct caacggcaaa 961gaatacaaat gcaaagtgtc caacaaagca ctcccagccc ctatcgagaa gactattagt 1021aaggcaaagg ggcagcctcg tgaaccacag gtgtacactc tgccacccag tagagaggaa 1081atgacaaaga accaagtctc attgacctgc ctggtgaaag gcttctaccc cagcgacatc 1141gccgttgagt gggagagtaa cggtcagcct gagaacaatt acaagacaac ccccccagtg 1201ctggatagtg acgggtcttt ctttctgtac agtaagctga ctgtggacaa gtcccgctgg 1261cagcagggta acgtcttcag ctgttccgtg atgcacgagg cattgcacaa ccactacacc 1321cagaagtcac tgagcctgag cccagggaag

Protein Sequence Defining the Full Length Ch4F11 Chimeric Heavy Chain(Mouse Heavy Chain Variable Region and Human IgG1 Constant Region) (SEQID NO:74)

1 evqlvesggg lvkpggslkl scaasgfafs sydmswvrqt pekrlewvay isrgggstyy 61pdtvkgrfti srdnakntly lqmsslksed tamyycgrha ttaywyfdvw gagttvtvss 121astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss 181glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg 241psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn 301styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree 361mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 421qqgnvfscsv mhealhnhyt qkslslspgk

Nucleic Acid Sequence Encoding the Full Length Sh4F11 Hv3-23 Heavy Chain(Humanized Heavy Chain Variable Region and Human IgG1 Constant Region)(SEQ ID NO:75)

1 gaagtacagt tgttggagtc aggaggaggg ttggtccagc cgggtggatc gttgcggctt 61tcgtgtgcgg cgtcgggatt cgcgttttca tcgtatgaca tgtcgtgggt gaggcaggca 121ccggggaaag ggcttgaatg ggtatcgtac atttcgagag ggggaggatc gacgtattac 181ccggatacgg tgaaaggaag gtttacgatc tcgcgcgaca attcaaagaa tacgctttat 241cttcagatga actcgctccg agcggaagat acggcggtat actattgcgg tcgccatgcg 301acgacggcgt attggtattt cgatgtgtgg ggacaaggga cgatggtcac ggtgtcgtcg 361gcctcaacaa aaggaccaag tgtgttccca ctcgccccta gcagcaagag tacatccggg 421ggcactgcag cactcggctg cctcgtcaag gattattttc cagagccagt aaccgtgagc 481tggaacagtg gagcactcac ttctggtgtc catacttttc ctgctgtcct gcaaagctct 541ggcctgtact cactcagctc cgtcgtgacc gtgccatctt catctctggg cactcagacc 601tacatctgta atgtaaacca caagcctagc aatactaagg tcgataagcg ggtggaaccc 661aagagctgcg acaagactca cacttgtccc ccatgccctg cccctgaact tctgggcggt 721cccagcgtct ttttgttccc accaaagcct aaagatactc tgatgataag tagaacaccc 781gaggtgacat gtgttgttgt agacgtttcc cacgaggacc cagaggttaa gttcaactgg 841tacgttgatg gagtcgaagt acataatgct aagaccaagc ctagagagga gcagtataat 901agtacatacc gtgtagtcag tgttctcaca gtgctgcacc aagactggct caacggcaaa 961gaatacaaat gcaaagtgtc caacaaagca ctcccagccc ctatcgagaa gactattagt 1021aaggcaaagg ggcagcctcg tgaaccacag gtgtacactc tgccacccag tagagaggaa 1081atgacaaaga accaagtctc attgacctgc ctggtgaaag gcttctaccc cagcgacatc 1141gccgttgagt gggagagtaa cggtcagcct gagaacaatt acaagacaac ccccccagtg 1201ctggatagtg acgggtcttt ctttctgtac agtaagctga ctgtggacaa gtcccgctgg 1261cagcagggta acgtcttcag ctgttccgtg atgcacgagg cattgcacaa ccactacacc 1321cagaagtcac tgagcctgag cccagggaag

Protein Sequence Defining the Full Length Sh4F11 Hv3-23 Heavy Chain(Humanized Heavy Chain Variable Region and Human IgG1 Constant Region)(SEQ ID NO:76)

1 evqllesggg lvqpggslrl scaasgfafs sydmswvrqa pgkglewvsy isrgggstyy 61pdtvkgrfti srdnskntly lqmnslraed tavyycgrha ttaywyfdvw gqgtmvtvss 121astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss 181glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg 241psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn 301styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree 361mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 421qqgnvfscsv mhealhnhyt qkslslspgk

Nucleic Acid Sequence Encoding the Full Length Sh4F11 Hv3-23 A28T S31HT62S Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1Constant Region) (SEQ ID NO:77)

1 gaagtacagt tgttggagtc aggaggaggg ttggtccagc cgggtggatc gttgcggctt 61tcgtgtgcgg cgtcgggatt caccttttca cactatgaca tgtcgtgggt gaggcaggca 121ccggggaaag ggcttgaatg ggtatcgtac atttcgagag ggggaggatc gacgtattac 181ccggattccg tgaaaggaag gtttacgatc tcgcgcgaca attcaaagaa tacgctttat 241cttcagatga actcgctccg agcggaagat acggcggtat actattgcgg tcgccatgcg 301acgacggcgt attggtattt cgatgtgtgg ggacaaggga cgatggtcac ggtgtcgtcg 361gcctcaacaa aaggaccaag tgtgttccca ctcgccccta gcagcaagag tacatccggg 421ggcactgcag cactcggctg cctcgtcaag gattattttc cagagccagt aaccgtgagc 481tggaacagtg gagcactcac ttctggtgtc catacttttc ctgctgtcct gcaaagctct 541ggcctgtact cactcagctc cgtcgtgacc gtgccatctt catctctggg cactcagacc 601tacatctgta atgtaaacca caagcctagc aatactaagg tcgataagcg ggtggaaccc 661aagagctgcg acaagactca cacttgtccc ccatgccctg cccctgaact tctgggcggt 721cccagcgtct ttttgttccc accaaagcct aaagatactc tgatgataag tagaacaccc 781gaggtgacat gtgttgttgt agacgtttcc cacgaggacc cagaggttaa gttcaactgg 841tacgttgatg gagtcgaagt acataatgct aagaccaagc ctagagagga gcagtataat 901agtacatacc gtgtagtcag tgttctcaca gtgctgcacc aagactggct caacggcaaa 961gaatacaaat gcaaagtgtc caacaaagca ctcccagccc ctatcgagaa gactattagt 1021aaggcaaagg ggcagcctcg tgaaccacag gtgtacactc tgccacccag tagagaggaa 1081atgacaaaga accaagtctc attgacctgc ctggtgaaag gcttctaccc cagcgacatc 1141gccgttgagt gggagagtaa cggtcagcct gagaacaatt acaagacaac ccccccagtg 1201ctggatagtg acgggtcttt ctttctgtac agtaagctga ctgtggacaa gtcccgctgg 1261cagcagggta acgtcttcag ctgttccgtg atgcacgagg cattgcacaa ccactacacc 1321cagaagtcac tgagcctgag cccagggaag

Protein Sequence Defining the Full Length Sh4F11 Hv3-23 A28T S31H T62SHeavy Chain (Humanized Heavy Chain Variable Region and Human IgG1Constant Region) (SEQ ID NO:78)

1 evqllesggg lvqpggslrl scaasgftfs hydmswvrqa pgkglewvsy isrgggstyy 61pdsvkgrfti srdnskntly lqmnslraed tavyycgrha ttaywyfdvw gqgtmvtvss 121astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss 181glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg 241psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn 301styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree 361mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 421qqgnvfscsv mhealhnhyt qkslslspgk

Nucleic Acid Sequence Encoding the Full Length Sh4F11 Hv3-23 S31H T62SHeavy Chain (Humanized Heavy Chain Variable Region and Human IgG1Constant Region) (SEQ ID NO:79)

1 gaagtacagt tgttggagtc aggaggaggg ttggtccagc cgggtggatc gttgcggctt 61tcgtgtgcgg cgtcgggatt cgcgttttca cactatgaca tgtcgtgggt gaggcaggca 121ccggggaaag ggcttgaatg ggtatcgtac atttcgagag ggggaggatc gacgtattac 181ccggattccg tgaaaggaag gtttacgatc tcgcgcgaca attcaaagaa tacgctttat 241cttcagatga actcgctccg agcggaagat acggcggtat actattgcgg tcgccatgcg 301acgacggcgt attggtattt cgatgtgtgg ggacaaggga cgatggtcac ggtgtcgtcg 361gcctcaacaa aaggaccaag tgtgttccca ctcgccccta gcagcaagag tacatccggg 421ggcactgcag cactcggctg cctcgtcaag gattattttc cagagccagt aaccgtgagc 481tggaacagtg gagcactcac ttctggtgtc catacttttc ctgctgtcct gcaaagctct 541ggcctgtact cactcagctc cgtcgtgacc gtgccatctt catctctggg cactcagacc 601tacatctgta atgtaaacca caagcctagc aatactaagg tcgataagcg ggtggaaccc 661aagagctgcg acaagactca cacttgtccc ccatgccctg cccctgaact tctgggcggt 721cccagcgtct ttttgttccc accaaagcct aaagatactc tgatgataag tagaacaccc 781gaggtgacat gtgttgttgt agacgtttcc cacgaggacc cagaggttaa gttcaactgg 841tacgttgatg gagtcgaagt acataatgct aagaccaagc ctagagagga gcagtataat 901agtacatacc gtgtagtcag tgttctcaca gtgctgcacc aagactggct caacggcaaa 961gaatacaaat gcaaagtgtc caacaaagca ctcccagccc ctatcgagaa gactattagt 1021aaggcaaagg ggcagcctcg tgaaccacag gtgtacactc tgccacccag tagagaggaa 1081atgacaaaga accaagtctc attgacctgc ctggtgaaag gcttctaccc cagcgacatc 1141gccgttgagt gggagagtaa cggtcagcct gagaacaatt acaagacaac ccccccagtg 1201ctggatagtg acgggtcttt ctttctgtac agtaagctga ctgtggacaa gtcccgctgg 1261cagcagggta acgtcttcag ctgttccgtg atgcacgagg cattgcacaa ccactacacc 1321cagaagtcac tgagcctgag cccagggaag

Protein Sequence Defining the Full Length Sh4F11 Hv3-23 S31H T62S HeavyChain (Humanized Heavy Chain Variable Region and Human IgG1 ConstantRegion) (SEQ ID NO:80)

1 evqllesggg lvqpggslrl scaasgfafs hydmswvrqa pgkglewvsy isrgggstyy 61pdsvkgrfti srdnskntly lqmnslraed tavyycgrha ttaywyfdvw gqgtmvtvss 121astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss 181glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg 241psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn 301styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree 361mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 421qqgnvfscsv mhealhnhyt qkslslspgk

Nucleic Acid Sequence Encoding the Full Length Sh4F11 Hv3-23 A28T S31NT62S Heavy Chain (Humanized Heavy Chain Variable Region and Human IgG1Constant Region) (SEQ ID NO:81)

1 gaagtacagt tgttggagtc aggaggaggg ttggtccagc cgggtggatc gttgcggctt 61tcgtgtgcgg cgtcgggatt caccttttca aactatgaca tgtcgtgggt gaggcaggca 121ccggggaaag ggcttgaatg ggtatcgtac atttcgagag ggggaggatc gacgtattac 181ccggattccg tgaaaggaag gtttacgatc tcgcgcgaca attcaaagaa tacgctttat 241cttcagatga actcgctccg agcggaagat acggcggtat actattgcgg tcgccatgcg 301acgacggcgt attggtattt cgatgtgtgg ggacaaggga cgatggtcac ggtgtcgtcg 361gcctcaacaa aaggaccaag tgtgttccca ctcgccccta gcagcaagag tacatccggg 421ggcactgcag cactcggctg cctcgtcaag gattattttc cagagccagt aaccgtgagc 481tggaacagtg gagcactcac ttctggtgtc catacttttc ctgctgtcct gcaaagctct 541ggcctgtact cactcagctc cgtcgtgacc gtgccatctt catctctggg cactcagacc 601tacatctgta atgtaaacca caagcctagc aatactaagg tcgataagcg ggtggaaccc 661aagagctgcg acaagactca cacttgtccc ccatgccctg cccctgaact tctgggcggt 721cccagcgtct ttttgttccc accaaagcct aaagatactc tgatgataag tagaacaccc 781gaggtgacat gtgttgttgt agacgtttcc cacgaggacc cagaggttaa gttcaactgg 841tacgttgatg gagtcgaagt acataatgct aagaccaagc ctagagagga gcagtataat 901agtacatacc gtgtagtcag tgttctcaca gtgctgcacc aagactggct caacggcaaa 961gaatacaaat gcaaagtgtc caacaaagca ctcccagccc ctatcgagaa gactattagt 1021aaggcaaagg ggcagcctcg tgaaccacag gtgtacactc tgccacccag tagagaggaa 1081atgacaaaga accaagtctc attgacctgc ctggtgaaag gcttctaccc cagcgacatc 1141gccgttgagt gggagagtaa cggtcagcct gagaacaatt acaagacaac ccccccagtg 1201ctggatagtg acgggtcttt ctttctgtac agtaagctga ctgtggacaa gtcccgctgg 1261cagcagggta acgtcttcag ctgttccgtg atgcacgagg cattgcacaa ccactacacc 1321cagaagtcac tgagcctgag cccagggaag

Protein Sequence Defining the Full Length Sh4F11 A28T S31N T62S HeavyChain (Humanized Heavy Chain Variable Region and Human IgG1 ConstantRegion) (SEQ ID NO:82)

1 evqllesggg lvqpggslrl scaasgftfs nydmswvrqa pgkglewvsy isrgggstyy 61pdsvkgrfti srdnskntly lqmnslraed tavyycgrha ttaywyfdvw gqgtmvtvss 121astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss 181glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg 241psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn 301styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree 361mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 421qqgnvfscsv mhealhnhyt qkslslspgk

Nucleic Acid Sequence Encoding the Full Length Sh4F11 Hv3-23 A28T T62SHeavy Chain (Humanized Heavy Chain Variable Region and Human IgG1Constant Region) (SEQ ID NO:83)

1 gaagtacagt tgttggagtc aggaggaggg ttggtccagc cgggtggatc gttgcggctt 61tcgtgtgcgg cgtcgggatt caccttttca tcgtatgaca tgtcgtgggt gaggcaggca 121ccggggaaag ggcttgaatg ggtatcgtac atttcgagag ggggaggatc gacgtattac 181ccggattccg tgaaaggaag gtttacgatc tcgcgcgaca attcaaagaa tacgctttat 241cttcagatga actcgctccg agcggaagat acggcggtat actattgcgg tcgccatgcg 301acgacggcgt attggtattt cgatgtgtgg ggacaaggga cgatggtcac ggtgtcgtcg 361gcctcaacaa aaggaccaag tgtgttccca ctcgccccta gcagcaagag tacatccggg 421ggcactgcag cactcggctg cctcgtcaag gattattttc cagagccagt aaccgtgagc 481tggaacagtg gagcactcac ttctggtgtc catacttttc ctgctgtcct gcaaagctct 541ggcctgtact cactcagctc cgtcgtgacc gtgccatctt catctctggg cactcagacc 601tacatctgta atgtaaacca caagcctagc aatactaagg tcgataagcg ggtggaaccc 661aagagctgcg acaagactca cacttgtccc ccatgccctg cccctgaact tctgggcggt 721cccagcgtct ttttgttccc accaaagcct aaagatactc tgatgataag tagaacaccc 781gaggtgacat gtgttgttgt agacgtttcc cacgaggacc cagaggttaa gttcaactgg 841tacgttgatg gagtcgaagt acataatgct aagaccaagc ctagagagga gcagtataat 901agtacatacc gtgtagtcag tgttctcaca gtgctgcacc aagactggct caacggcaaa 961gaatacaaat gcaaagtgtc caacaaagca ctcccagccc ctatcgagaa gactattagt 1021aaggcaaagg ggcagcctcg tgaaccacag gtgtacactc tgccacccag tagagaggaa 1081atgacaaaga accaagtctc attgacctgc ctggtgaaag gcttctaccc cagcgacatc 1141gccgttgagt gggagagtaa cggtcagcct gagaacaatt acaagacaac ccccccagtg 1201ctggatagtg acgggtcttt ctttctgtac agtaagctga ctgtggacaa gtcccgctgg 1261cagcagggta acgtcttcag ctgttccgtg atgcacgagg cattgcacaa ccactacacc 1321cagaagtcac tgagcctgag cccagggaag

Protein Sequence Defining the Full Length Sh4F11 Hv3-23 A28T T62SHeavyChain (Humanized Heavy Chain Variable Region and Human IgG1 ConstantRegion) (SEQ ID NO:84)

1 evqllesggg lvqpggslrl scaasgftfs sydmswvrqa pgkglewvsy isrgggstyy 61pdsvkgrfti srdnskntly lqmnslraed tavyycgrha ttaywyfdvw gqgtmvtvss 121astkgpsvfp lapsskstsg gtaalgclvk dyfpepvtvs wnsgaltsgv htfpavlqss 181glyslssvvt vpssslgtqt yicnvnhkps ntkvdkrvep kscdkthtcp pcpapellgg 241psvflfppkp kdtlmisrtp evtcvvvdvs hedpevkfnw yvdgvevhna ktkpreeqyn 301styrvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsree 361mtknqvsltc lvkgfypsdi avewesngqp ennykttppv ldsdgsffly skltvdksrw 421qqgnvfscsv mhealhnhyt qkslslspgk

Nucleic Acid Sequence Encoding the Full Length Ch4F11 Chimeric LightChain (Mouse Kappa Chain Variable Region and Human Kappa ConstantRegion) (SEQ ID NO:85)

1 gacgtggtaa tgacgcagac gccgttgtcc cttcctgtct cgctcggaga tcaggcgtcg 61atctcgtgta gaagctcgca gtcactcgtc cataccaacg ggaatacata tcttcactgg 121tatttgcaaa agcccggaca gtcaccgaag ctcttgatct acaaagtatc caatcggttt 181tcgggggtgc ccgaccgatt ctcgggatcg ggttcgggga cggattttac gttgaagatt 241tcgcgggtgg aagcggagga tctcggtgtc tacttttgtt cgcagtcaac gcatgtcccg 301tggacgttcg gaggcgggac aaaacttgag atcaagcgca cagttgctgc ccccagcgtg 361ttcattttcc cacctagcga tgagcagctg aaaagcggta ctgcctctgt cgtatgcttg 421ctcaacaact tttacccacg tgaggctaag gtgcagtgga aagtggataa tgcacttcaa 481tctggaaaca gtcaagagtc cgtgacagaa caggacagca aagactcaac ttattcactc 541tcttccaccc tgactctgtc caaggcagac tatgaaaaac acaaggtata cgcctgcgag 601gttacacacc agggtttgtc tagtcctgtc accaagtcct tcaatagggg cgaatgt

Protein Sequence Defining the Full Length Ch4F11 Chimeric Light Chain(Mouse Kappa Chain Variable Region and Human Kappa Constant Region) (SEQID NO:86)

1 dvvmtqtpls lpvslgdqas iscrssqslv htngntylhw ylqkpgqspk lliykvsnrf 61sgvpdrfsgs gsgtdftlki srveaedlgv yfcsqsthvp wtfgggtkle ikrtvaapsv 121fifppsdeql ksgtasvvcl lnnfypreak vqwkvdnalq sgnsqesvte qdskdstysl 181sstltlskad yekhkvyace vthqglsspv tksfnrgec

Nucleic Acid Sequence Encoding the Full Length Hu4F11 Kv2D-29 LightChain (Humanized Kappa Chain Variable Region and Human Kappa ConstantRegion (Version 1)) (SEQ ID NO:87)

1 gatgtagtca tgacccaaac gccgctttcg ttgtcggtga cgcccggaca gcccgcgtca 61atctcgtgtc ggtcatcgca gtcgttggta cacacaaacg gtaatacgta tctccattgg 121tatctccaga agcccggcca gtcgccgcag ctcttgatct acaaagtgag caatcgcttt 181tcgggggtgc cggatcggtt ctcgggatcg gggtcaggaa cggacttcac gcttaagatt 241tcgagggtcg aggcggagga tgtcggagtc tacttttgtt cgcagtccac acatgtcccc 301tggacgtttg ggcaggggac gaaggtggaa atcaagcgca cagttgctgc ccccagcgtg 361ttcattttcc cacctagcga tgagcagctg aaaagcggta ctgcctctgt cgtatgcttg 421ctcaacaact tttacccacg tgaggctaag gtgcagtgga aagtggataa tgcacttcaa 481tctggaaaca gtcaagagtc cgtgacagaa caggacagca aagactcaac ttattcactc 541tcttccaccc tgactctgtc caaggcagac tatgaaaaac acaaggtata cgcctgcgag 601gttacacacc agggtttgtc tagtcctgtc accaagtcct tcaatagggg cgaatgt

Protein Sequence Defining the Full Length Hu4F11 Kv2D-29 Light Chain(Humanized Kappa Chain Variable Region and Human Kappa Constant Region)(SEQ ID NO:88)

1 dvvmtqtpls lsvtpgqpas iscrssqslv htngntylhw ylqkpgqspq lliykvsnrf 61sgvpdrfsgs gsgtdftlki srveaedvgv yfcsqsthvp wtfgqgtkve ikrtvaapsv 121fifppsdeql ksgtasvvcl lnnfypreak vqwkvdnalq sgnsqesvte qdskdstysl 181sstltlskad yekhkvyace vthqglsspv tksfnrgec

Nucleic Acid Sequence Encoding the Full Length Hu4F11 Kv2D-29 N28H LightChain (Humanized Kappa Chain Variable Region and Human Kappa ConstantRegion (Version 2)) (SEQ ID NO:89)

1 gatgtagtca tgacccaaac gccgctttcg ttgtcggtga cgcccggaca gcccgcgtca 61atctcgtgtc ggtcatcgca gtcgttggta cacacacacg gtaatacgta tctccattgg 121tatctccaga agcccggcca gtcgccgcag ctcttgatct acaaagtgag caatcgcttt 181tcgggggtgc cggatcggtt ctcgggatcg gggtcaggaa cggacttcac gcttaagatt 241tcgagggtcg aggcggagga tgtcggagtc tacttttgtt cgcagtccac acatgtcccc 301tggacgtttg ggcaggggac gaaggtggaa atcaagcgca cagttgcagc ccccagcgtg 361ttcattttcc cacctagcga tgagcagctg aaaagcggta ctgcctctgt cgtatgcttg 421ctcaacaact tttacccacg tgaggctaag gtgcagtgga aagtggataa tgcacttcaa 481tctggaaaca gtcaagagtc cgtgacagaa caggacagca aagactcaac ttattcactc 541tcttccaccc tgactctgtc caaggcagac tatgaaaaac acaaggtata cgcctgcgag 601gttacacacc agggtttgtc tagtcctgtc accaagtcct tcaatagggg cgaatgt

Protein Sequence Defining the Full Length Hu4F11 Kv2D-29 N28H LightChain (Humanized Kappa Chain Variable Region and Human Kappa ConstantRegion) (SEQ ID NO:90)

1 dvvmtqtpls lsvtpgqpas iscrssqslv hthgntylhw ylqkpgqspq lliykvsnrf 61sgvpdrfsgs gsgtdftlki srveaedvgv yfcsqsthvp wtfgqgtkve ikrtvaapsv 121fifppsdeql ksgtasvvcl lnnfypreak vqwkvdnalq sgnsqesvte qdskdstysl 181sstltlskad yekhkvyace vthqglsspv tksfnrgec

Nucleic Acid Sequence Encoding the Full Length Hu4F11 Kv2D-29 N28Q LightChain (Humanized Kappa Chain Variable Region and Human Kappa ConstantRegion (Version 2)) (SEQ ID NO:91)

1 gatgtagtca tgacccaaac gccgctttcg ttgtcggtga cgcccggaca gcccgcgtca 61atctcgtgtc ggtcatcgca gtcgttggta cacacacaag gtaatacgta tctccattgg 121tatctccaga agcccggcca gtcgccgcag ctcttgatct acaaagtgag caatcgcttt 181tcgggggtgc cggatcggtt ctcgggatcg gggtcaggaa cggacttcac gcttaagatt 241tcgagggtcg aggcggagga tgtcggagtc tacttttgtt cgcagtccac acatgtcccc 301tggacgtttg ggcaggggac gaaggtggaa atcaagcgca cagttgcagc ccccagcgtg 361ttcattttcc cacctagcga tgagcagctg aaaagcggta ctgcctctgt cgtatgcttg 421ctcaacaact tttacccacg tgaggctaag gtgcagtgga aagtggataa tgcacttcaa 481tctggaaaca gtcaagagtc cgtgacagaa caggacagca aagactcaac ttattcactc 541tcttccaccc tgactctgtc caaggcagac tatgaaaaac acaaggtata cgcctgcgag 601gttacacacc agggtttgtc tagtcctgtc accaagtcct tcaatagggg cgaatgt

Protein Sequence Defining the Full Length Hu4F11 Kv2D-29 N28Q LightChain (Humanized Kappa Chain Variable Region and Human Kappa ConstantRegion) (SEQ ID NO:92)

1 dvvmtqtpls lsvtpgqpas iscrssqslv htqgntylhw ylqkpgqspq lliykvsnrf 61sgvpdrfsgs gsgtdftlki srveaedvgv yfcsqsthvp wtfgqgtkve ikrtvaapsv 121fifppsdeql ksgtasvvcl lnnfypreak vqwkvdnalq sgnsqesvte qdskdstysl 181sstltlskad yekhkvyace vthqglsspv tksfnrgec

Nucleic Acid Sequence Encoding the Full Length Hu4F11 Kv2D-29 N28Y LightChain (Humanized Kappa Chain Variable Region and Human Kappa ConstantRegion (Version 2)) (SEQ ID NO:93)

1 gatgtagtca tgacccaaac gccgctttcg ttgtcggtga cgcccggaca gcccgcgtca 61atctcgtgtc ggtcatcgca gtcgttggta cacacatacg gtaatacgta tctccattgg 121tatctccaga agcccggcca gtcgccgcag ctcttgatct acaaagtgag caatcgcttt 181tcgggggtgc cggatcggtt ctcgggatcg gggtcaggaa cggacttcac gcttaagatt 241tcgagggtcg aggcggagga tgtcggagtc tacttttgtt cgcagtccac acatgtcccc 301tggacgtttg ggcaggggac gaaggtggaa atcaagcgca cagttgcagc ccccagcgtg 361ttcattttcc cacctagcga tgagcagctg aaaagcggta ctgcctctgt cgtatgcttg 421ctcaacaact tttacccacg tgaggctaag gtgcagtgga aagtggataa tgcacttcaa 481tctggaaaca gtcaagagtc cgtgacagaa caggacagca aagactcaac ttattcactc 541tcttccaccc tgactctgtc caaggcagac tatgaaaaac acaaggtata cgcctgcgag 601gttacacacc agggtttgtc tagtcctgtc accaagtcct tcaatagggg cgaatgt

Protein Sequence Defining the Full Length Hu4F11 Kv2D-29 N28Y LightChain (Humanized Kappa Chain Variable Region and Human Kappa ConstantRegion) (SEQ ID NO:94)

1 dvvmtqtpls lsvtpgqpas iscrssqslv htygntylhw ylqkpgqspq lliykvsnrf 61sgvpdrfsgs gsgtdftlki srveaedvgv yfcsqsthvp wtfgqgtkve ikrtvaapsv 121fifppsdeql ksgtasvvcl lnnfypreak vqwkvdnalq sgnsqesvte qdskdstysl 181sstltlskad yekhkvyace vthqglsspv tksfnrgec

Table 12 is a concordance chart showing the SEQ ID NO. of each sequencediscussed in this Example.

TABLE 12 SEQ ID NO. Nucleic Acid or Protein 68 Human IgG1constant-nucleic acid 69 Human IgG1 constant-protein 70 Human Kappaconstant-nucleic acid (version 1) 71 Human Kappa constant-nucleic acid(version 2) 72 Human Kappa constant-protein (nucleic acid versions 1 and2 encode the same amino acid sequence) 73 Chimeric Ch4F11 HeavyVariable + Human IgG1 constant-nucleic acid 74 Chimeric Ch4F11 HeavyVariable + Human IgG1 constant-protein 75 Humanized Sh4F11 Hv3-23 HeavyHuman Variable + Human IgG1 constant-nucleic acid 76 Humanized Sh4F11Hv3-23 Heavy Human Variable + Human IgG1 constant-protein 77 HumanizedSh4F11 Hv3-23 A28T S31H T62S Heavy Human Variable + Human IgG1constant-nucleic acid 78 Humanized Sh4F11 Hv3-23 A28T S31H T62S HeavyHuman Variable + Human IgG1 constant-protein 79 Humanized Sh4F11 Hv3-23S31H T62S Heavy Human Variable + Human IgG1 constant - nucleic acid 80Humanized Sh4F11 Hv3-23 S31H T62S Heavy Human Variable + Human IgG1constant - protein 81 Humanized Sh4F11 Hv3-23 A28T S31N T62S Heavy HumanVariable + Human IgG1 constant-nucleic acid 82 Humanized Sh4F11 Hv3-23A28T S31N T62S Heavy Human Variable + Human IgG1 constant-protein 83Humanized Sh4F11 Hv3-23 A28T T62S Heavy Human Variable + Human IgG1constant - nucleic acid 84 Humanized Sh4F11 Hv3-23 A28T T62S Heavy HumanVariable + Human IgG1 constant - protein 85 Chimeric Ch4F11 KappaVariable + Human Kappa constant-nucleic acid (version 1) 86 ChimericCh4F11 Kappa Variable + Human Kappa constant-protein 87 Humanized Hu4F11Kv2D-29 Kappa Human Variable + Human Kappa constant-nucleic acid(version 1) 88 Humanized Hu4F11 Kv2D-29 Kappa Human Variable + HumanKappa constant-protein 89 Humanized Hu4F11 Kv2D-29 N28H Kappa HumanVariable + Human Kappa constant - nucleic acid (version 2) 90 HumanizedHu4F11 Kv2D-29 N28H Kappa Human Variable + Human Kappa constant -protein 91 Humanized Hu4F11 Kv2D-29 N28Q Kappa Human Variable + HumanKappa constant - nucleic acid (version 2) 92 Humanized Hu4F11 Kv2D-29N28Q Kappa Human Variable + Human Kappa constant - protein 93 HumanizedHu4F11 Kv2D-29 N28Y Kappa Human Variable + Human Kappa constant -nucleic acid (version 2) 94 Humanized Hu4F11 Kv2D-29 N28Y Kappa HumanVariable + Human Kappa constant - protein

Table 13 below shows antibodies containing chimeric immunoglobulin heavyand light chains and exemplary combinations of the full-length chimericor humanized immunoglobulin heavy and light chains.

TABLE 13 Antibody Name Heavy Chain Light Chain Hu4F11-1 Ch4F11 ChimericHeavy Variable + Ch4F11 Chimeric Kappa Variable + Human IgG1 constantHuman Kappa constant (SEQ ID NO: 74) (SEQ ID NO: 86) Hu4F11-10 HumanizedSh4F11 Hv3-23 Heavy Ch4F11 Chimeric Kappa Variable + Human Variable +Human IgG1 constant Human Kappa constant (SEQ ID NO: 76) (SEQ ID NO: 86)Hu4F11-18 Ch4F11 Chimeric Heavy Variable + Humanized Hu4F11 Kv2D-29Human Human IgG1 constant Variable + Human Kappa constant (SEQ ID NO:74) (SEQ ID NO: 88) Hu4F11-32 Humanized Sh4F11 Hv3-23 Heavy HumanizedHu4F11 Kv2D-29 Human Human Variable + Human IgG1 constant KappaVariable + Human Kappa (SEQ ID NO: 76) constant (SEQ ID NO: 88)Hu4F11-69 Humanized Sh4F11 Hv3-23 A28T S31N Humanized Hu4F11 Kv2D-29N28H T62S Heavy Human Variable + Human Human Kappa Variable + HumanKappa IgG1 constant constant (SEQ ID NO: 82) (SEQ ID NO: 90) Hu4F11-70Humanized Sh4F11 Hv3-23 A28T S31H Humanized Hu4F11 Kv2D-29 N28H T62SHeavy Human Variable + Human Human Kappa Variable + Human Kappa IgG1constant constant (SEQ ID NO: 78) (SEQ ID NO: 90) Hu4F11-71 HumanizedSh4F11 Hv3-23 A28T T62S Humanized Hu4F11 Kv2D-29 N28H Heavy HumanVariable + Human IgG1 Human Kappa Variable + Human Kappa constantconstant (SEQ ID NO: 84) (SEQ ID NO: 90) Hu4F11-72 Humanized Sh4F11Hv3-23 S31H T62S Humanized Hu4F11 Kv2D-29 N28H Heavy Human Variable +Human IgG1 Human Kappa Variable + Human Kappa constant constant (SEQ IDNO: 80) (SEQ ID NO: 90) Hu4F11-73 Humanized Sh4F11 Hv3-23 A28T S31NHumanized Hu4F11 Kv2D-29 N28Y T62S Heavy Human Variable + Human HumanKappa Variable + Human Kappa IgG1 constant constant (SEQ ID NO: 82) (SEQID NO: 94) Hu4F11-74 Humanized Sh4F11 Hv3-23 A28T S31H Humanized Hu4F11Kv2D-29 N28Y T62S Heavy Human Variable + Human Human Kappa Variable +Human Kappa IgG1 constant constant (SEQ ID NO: 78) (SEQ ID NO: 94)Hu4F11-75 Humanized Sh4F11 Hv3-23 A28T T62S Humanized Hu4F11 Kv2D-29N28Y Heavy Human Variable + Human IgG1 Human Kappa Variable + HumanKappa constant constant (SEQ ID NO: 84) (SEQ ID NO: 94) Hu4F11-76Humanized Sh4F11 Hv3-23 S31H T62S Humanized Hu4F11 Kv2D-29 N28Y HeavyHuman Variable + Human IgG1 Human Kappa Variable + Human Kappa constantconstant (SEQ ID NO: 80) (SEQ ID NO: 94) Hu4F11-77 Humanized Sh4F11Hv3-23 A28T S31N Humanized Hu4F11 Kv2D-29 N28Q T62S Heavy HumanVariable + Human Human Kappa Variable + Human Kappa IgG1 constantconstant (SEQ ID NO: 82) (SEQ ID NO: 92) Hu4F11-78 Humanized Sh4F11Hv3-23 A28T S31H Humanized Hu4F11 Kv2D-29 N28Q T62S Heavy HumanVariable + Human Human Kappa Variable + Human Kappa IgG1 constantconstant (SEQ ID NO: 78) (SEQ ID NO: 92) Hu4F11-79 Humanized Sh4F11Hv3-23 A28T T62S Humanized Hu4F11 Kv2D-29 N28Q Heavy Human Variable +Human IgG1 Human Kappa Variable + Human Kappa constant constant (SEQ IDNO: 84) (SEQ ID NO: 92) Hu4F11-80 Humanized Sh4F11 Hv3-23 S31H T62SHumanized Hu4F11 Kv2D-29 N28Q Heavy Human Variable + Human IgG1 HumanKappa Variable + Human Kappa constant constant (SEQ ID NO: 80) (SEQ IDNO: 92)

The antibody construct containing the full length chimeric heavy andlight chains is designated below:

-   -   Chimeric 4F11 (Hu4F11-1)=Full Length Ch4F11 Chimeric Heavy Chain        (Mouse Heavy Chain Variable Region and Human IgG1 Constant        Region) (SEQ ID NO:74) plus Full Length Ch4F11 Chimeric Light        Chain (Mouse Kappa Chain Variable Region and Human Kappa        Constant Region) (SEQ ID NO:86)

Thirteen of the possible antibody constructs containing the full lengthimmunoglobulin heavy and light chains containing humanized variableregions are designated below:

-   -   Hu4F11-32=Full Length Sh4F11 Hv3-23 Heavy Chain (Humanized Heavy        Chain Variable Region and Human IgG1 Constant Region) (SEQ ID        NO:76) plus Full Length Hu4F11 Kv2D-29 Light Chain (Humanized        Kappa Chain Variable Region and Human Kappa Constant Region)        (SEQ ID NO:88)    -   Hu4F11-69=Full Length Sh4F11 Hv3-23 A28T S31N T625 Heavy Chain        (Humanized Heavy Chain Variable Region and Human IgG1 Constant        Region) (SEQ ID NO:82) plus Full Length Hu4F11 Kv2D-29 N28H        Light Chain (Humanized Kappa Chain Variable Region and Human        Kappa Constant Region) (SEQ ID NO:90)    -   Hu4F11-70=Full Length Sh4F11 Hv3-23 A28T S31H T62S Heavy Chain        (Humanized Heavy Chain Variable Region and Human IgG1 Constant        Region) (SEQ ID NO:78) plus Full Length Hu4F11 Kv2D-29 N28H        Light Chain (Humanized Kappa Chain Variable Region and Human        Kappa Constant Region) (SEQ ID NO:90)    -   Hu4F11-71=Full Length Sh4F11 Hv3-23 A28T T62S Heavy Chain        (Humanized Heavy Chain Variable Region and Human IgG1 Constant        Region) (SEQ ID NO:84) plus Full Length Hu4F11 Kv2D-29 N28H        Light Chain (Humanized Kappa Chain Variable Region and Human        Kappa Constant Region) (SEQ ID NO:90)    -   Hu4F11-72=Full Length Sh4F11 Hv3-23 S31H T62S Heavy Chain        (Humanized Heavy Chain Variable Region and Human IgG1 Constant        Region) (SEQ ID NO:80) plus Full Length Hu4F11 Kv2D-29 N28H        Light Chain (Humanized Kappa Chain Variable Region and Human        Kappa Constant Region) (SEQ ID NO:90)    -   Hu4F11-73=Full Length Sh4F11 Hv3-23 A28T S31N T625 Heavy Chain        (Humanized Heavy Chain Variable Region and Human IgG1 Constant        Region) (SEQ ID NO:82) plus Full Length Hu4F11 Kv2D-29 N28Y        Light Chain (Humanized Kappa Chain Variable Region and Human        Kappa Constant Region) (SEQ ID NO:94)    -   Hu4F11-74=Full Length Sh4F11 Hv3-23 A28T S31H T62S Heavy Chain        (Humanized Heavy Chain Variable Region and Human IgG1 Constant        Region) (SEQ ID NO:78) plus Full Length Hu4F11 Kv2D-29 N28Y        Light Chain (Humanized Kappa Chain Variable Region and Human        Kappa Constant Region) (SEQ ID NO:94)    -   Hu4F11-75=Full Length Sh4F11 Hv3-23 A28T T62S Heavy Chain        (Humanized Heavy Chain Variable Region and Human IgG1 Constant        Region) (SEQ ID NO:84) plus Full Length Hu4F11 Kv2D-29 N28Y        Light Chain (Humanized Kappa Chain Variable Region and Human        Kappa Constant Region) (SEQ ID NO:94)    -   Hu4F11-76=Full Length Sh4F11 Hv3-23 S31H T62S Heavy Chain        (Humanized Heavy Chain Variable Region and Human IgG1 Constant        Region) (SEQ ID NO:80) plus Full Length Hu4F11 Kv2D-29 N28Y        Light Chain (Humanized Kappa Chain Variable Region and Human        Kappa Constant Region) (SEQ ID NO:94)    -   Hu4F11-77=Full Length Sh4F11 Hv3-23 A28T S31N T625_Heavy Chain        (Humanized Heavy Chain Variable Region and Human IgG1 Constant        Region) (SEQ ID NO:82) plus Full Length Hu4F11 Kv2D-29 N28Q        Light Chain (Humanized Kappa Chain Variable Region and Human        Kappa Constant Region) (SEQ ID NO:92)    -   Hu4F11-78=Full Length Sh4F11 Hv3-23 A28T S31H T62S Heavy Chain        (Humanized Heavy Chain Variable Region and Human IgG1 Constant        Region) (SEQ ID NO:78) plus Full Length Hu4F11 Kv2D-29 N28Q        Light Chain (Humanized Kappa Chain Variable Region and Human        Kappa Constant Region) (SEQ ID NO:92)    -   Hu4F11-79=Full Length Sh4F11 Hv3-23 A28T T62S Heavy Chain        (Humanized Heavy Chain Variable Region and Human IgG1 Constant        Region) (SEQ ID NO:84) plus Full Length Hu4F11 Kv2D-29 N28Q        Light Chain (Humanized Kappa Chain Variable Region and Human        Kappa Constant Region) (SEQ ID NO:92)    -   Hu4F11-80=Full Length Sh4F11 Hv3-23 S31H T62S Heavy Chain        (Humanized Heavy Chain Variable Region and Human IgG1 Constant        Region) (SEQ ID NO:80) plus Full Length Hu4F11 Kv2D-29 N28Q        Light Chain (Humanized Kappa Chain Variable Region and Human        Kappa Constant Region) (SEQ ID NO:92)

Example 9: Binding Affinities of Humanized and Chimeric Anti-Notch3Monoclonal Antibodies

The binding affinities and kinetics of binding of chimeric and humanizedantibodies to monomeric recombinant human Notch3 extracellular domain(containing EGF like domains 1-11) fusion protein (monomeric rhNotch3)and monomeric recombinant cynomologus monkey Notch3 extracellular domain(containing EGF like domains 1-12) fusion protein (monomeric rcNotch3)were measured by surface plasmon resonance, using a BIAcore® T100instrument (GE Healthcare, Piscataway, N.J.).

Goat anti-human IgGs (Fc fragment specific, Jackson ImmunoResearch, WestGrove, Pa.) were immobilized on carboxymethylated dextran CM4 sensorchips by amine coupling, according to a standard protocol. Analyses wereperformed at 25 and 37° C. using PBS containing 0.05% surfactant P20 asrunning buffer. The antibodies were captured in individual flow cells ata flow rate of 10 μL/minute. Injection time was varied for each antibodyto yield an Rmax between 30 and 60 RU. Buffer or Notch3 protein dilutedin running buffer was injected sequentially over a reference surface (noantibody captured) and the active surface (antibody to be tested) for240 seconds at 60 μL/minute. The dissociation phase was monitored for upto 900 seconds. The surface was then regenerated with two 60-secondinjections of 10 mM Glycine-HCl, pH 2.25, at a flow rate of 30μL/minute. The Notch3 concentration range tested was 50 nM to 6.25 nM.

Kinetic parameters were determined using the kinetic function of theBIAevaluation software (GE Healthcare) with double referencesubtraction. Kinetic parameters for each antibody, k_(a) (associationrate constant), k_(d) (dissociation rate constant), and K_(D)(equilibrium dissociation constant) were determined. Kinetic values ofpurified monoclonal antibodies on monomeric rcNotch3 at 25° C. aresummarized in Table 14.

TABLE 14 Antibody k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) n Hu4F11-1 4.8E+051.0E−02 2.4E−08 5 Hu4F11-32 7.9E+05 7.5E−03 1.8E−08 8 Hu4F11-69 1.2E+059.4E−04 7.7E−09 1 Hu4F11-70 2.0E+05 1.0E−03 5.2E−09 1 Hu4F11-71 1.5E+064.0E−03 2.7E−09 1 Hu4F11-72 2.8E+06 4.5E−03 1.6E−09 1 Hu4F11-73 7.6E+051.9E−03 2.6E−09 1 Hu4F11-74 9.0E+05 2.5E−03 2.8E−09 1 Hu4F11-75 7.2E+052.6E−03 3.7E−09 1 Hu4F11-76 7.8E+05 1.9E−03 2.5E−09 1 Hu4F11-77 9.6E+052.5E−03 2.6E−09 1 Hu4F11-78 1.8E+06 3.8E−03 2.2E−09 1 Hu4F11-79 1.3E+064.6E−03 3.4E−09 1 Hu4F11-80 8.2E+05 3.2E−03 4.0E−09 1

Additional kinetic measurements were conducted for each antibody shownin Table 14. These measurements confirmed that the antibodies haveaffinities ranging from about 1 nM to about 25 nM for monomeric rcNotch3at 25° C.

Kinetic values of purified monoclonal antibodies on monomeric rcNotch3at 37° C. are summarized in Table 15.

TABLE 15 Antibody k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) n Hu4F11-32 5.0E+051.6E−02 3.2E−08 5 Hu4F11-69 4.0E+05 3.8E−03 1.1E−08 3 Hu4F11-70 3.7E+052.9E−03 9.1E−09 3 Hu4F11-71 4.1E+05 5.6E−03 1.4E−08 3 Hu4F11-72 4.4E+053.2E−03 9.8E−09 3 Hu4F11-73 5.0E+05 6.3E−03 1.5E−08 3 Hu4F11-74 4.4E+054.5E−03 1.2E−08 3 Hu4F11-75 3.8E+05 8.9E−03 2.4E−08 3 Hu4F11-76 5.4E+056.2E−03 1.2E−08 3 Hu4F11-77 9.1E+05 1.0E−02 1.2E−08 3 Hu4F11-78 6.6E+057.0E−03 1.1E−08 3 Hu4F11-79 9.8E+05 2.1E−02 2.3E−08 3 Hu4F11-80 9.1E+051.0E−02 1.2E−08 3

Additional kinetic measurements were conducted for each antibody shownin Table 15. These measurements confirmed that the antibodies haveaffinities ranging from about 9 nM to about 35 nM for monomeric rcNotch3at 37° C.

Kinetic values of purified monoclonal antibodies on monomeric rhNotch3at 37° C. are summarized in Table 16.

TABLE 16 Antibody k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) n Hu4F11-32 1.7E+064.6E−03 2.7E−09 1 Hu4F11-69 1.3E+06 3.8E−03 2.9E−09 1 Hu4F11-70 9.4E+052.8E−03 3.0E−09 1 Hu4F11-71 1.2E+06 3.7E−03 3.0E−09 1 Hu4F11-72 1.8E+059.4E−04 5.2E−09 1 Hu4F11-73 2.1E+05 1.3E−03 5.9E−09 1 Hu4F11-74 5.6E+052.2E−03 3.9E−09 1 Hu4F11-75 2.7E+06 9.0E−03 3.4E−09 1 Hu4F11-76 1.7E+065.5E−03 3.3E−09 1 Hu4F11-77 2.2E+06 7.3E−03 3.3E−09 1 Hu4F11-78 1.5E+065.5E−03 3.8E−09 1 Hu4F11-79 1.8E+06 6.0E−03 3.2E−09 1 Hu4F11-80 1.1E+064.1E−03 3.8E−09 1

Additional kinetic measurements were conducted for each antibody shownin Table 16. The additional measurements were averaged together withthose presented in Table 16. These combined measurements indicated thatthe antibodies have affinities ranging from about 7 nM to about 16 nMfor monomeric rhNotch3 at 37° C. (Table 17).

TABLE 17 Antibody k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) n Hu4F11-32 7.4E+068.4E−02 7.6E−09 2 Hu4F11-69 7.1E+05 3.7E−03 8.5E−09 3 Hu4F11-70 6.9E+053.9E−03 7.5E−09 4 Hu4F11-71 1.1E+06 9.5E−03 9.2E−09 3 Hu4F11-72 7.1E+056.9E−03 7.0E−09 4 Hu4F11-73 4.9E+05 4.6E−03 1.1E−08 3 Hu4F11-74 4.9E+055.1E−03 1.0E−08 4 Hu4F11-75 1.7E+06 1.3E−02 1.0E−08 3 Hu4F11-76 9.8E+056.4E−03 8.2E−09 3 Hu4F11-77 1.2E+06 7.4E−03 8.6E−09 3 Hu4F11-78 8.2E+056.0E−03 9.0E−09 4 Hu4F11-79 9.0E+05 9.1E−03 1.6E−08 3 Hu4F11-80 5.9E+055.6E−03 1.4E−08 3

The results in Tables 14-17 demonstrate that the chimeric and each ofthe humanized antibodies, have fast association rates (k_(a)), very slowdisassociation rates (k_(d)) and very high affinities (K_(D)). Inparticular, the antibodies have affinities ranging from about 1 nM toabout 35 nM.

Example 10: Binding of Humanized 4F11 Antibodies to CellSurface-Expressed Notch3

Binding of humanized 4F11 variants to cell surface Notch3 receptor wasconfirmed by FACS. FlpIn™ CHO lines stably expressing human Notch3, orFlpIn™ CHO lines expressing no human Notch receptors, were used forbinding experiments. CHO cells were harvested using Cell DissociationBuffer (Life Technologies, Grand Island, N.Y.) and resuspended at2.5×10⁶ cells/ml in PBS/0.5% BSA. 100 μl of cell suspension per samplewas added to a 96 well v-bottom plate. 4F11 humanized antibodies orhuman IgG were added at 5 μg/ml to the wells, mixed, and incubated onice for one hour. After washing with PBS/0.5% BSA, anti-human PEconjugated secondary antibody was added at a 1:100 dilution in PBS/0.5%BSA and allowed to incubate on ice in the dark for 30 minutes. Cellswere washed with PBS/0.5% BSA, then resuspended in 300 ul PBS/0.5% BSAand FACS analysis was performed. Expression of human Notch3 receptor wasconfirmed using anti-human Notch3 PE (BioLegend, San Diego, Calif.) as apositive control. FACS analysis confirmed that the humanized 4F11antibody variants bind to human Notch3 expressed on the cell surface.

Example 11: Inhibition of Ligand-Induced Notch3 ICD Cleavage byHumanized 4F11 Antibodies

Selected humanized 4F11 variants were tested for their ability toinhibit ligand-induced activation of Notch3, as measured by the presenceof cleaved ICD. The MDA-MB-468 breast cancer cell line, which expressesendogenous human Notch3, was plated in 96-well plates previously coatedwith hJag2-mFc fusion protein. The wells were prepared by dilutingα-mouse Fc (Jackson ImmunoResearch, West Grove, Pa.) to 5 μg/ml insterile-filtered carbonate-bicarbonate coating buffer, pH 9.4 (ThermoFisher Scientific, Rockland, Md.). Then 100 μl of the diluted antibodywas added to each well of a 96-well Maxisorp™ plate and incubatedovernight at 4° C. The next day, wells were washed three times withPBS/0.5% BSA before adding 100 μl of soluble Jag2-mFc fusion protein ormouse IgG Fc (Jackson Immunolabs) diluted to 5 μg/ml in PBS/0.5% BSA.After incubating for two hours at room temperature on an orbital shaker,the wells were washed three times with PBS/05% BSA to remove unboundligand. MDA-MB-468 cells were counted and resuspended in fresh growthmedia at 0.4×10⁶ cells/ml. Cells were pre-incubated with 10 ug/ml of the4F11 humanized antibodies for 30 minutes at 37° C., before seeding 100μl of the suspension into 96-well plates coated with hJag2-mFc ligand ormFc. Cells were incubated overnight at 37° C. The next day wells weregently washed with ice cold PBS, then cells harvested by adding RIPAbuffer containing protease inhibitors directly to the well. Lysates wereclarified by centrifugation in a refrigerated microcentrifuge.Supernatants were boiled with 5×SDS sample buffer before loading onto anSDS PAGE gel and Western blotting. Blots were probed with a α-Notch3antibody to detect the cleaved intracellular domain (Cell Signaling,Danvers, Mass.). The same blots were also probed with anti-β tubulin(Cell Signaling) for use as a loading control. Bands were quantitatedusing ImageLab software (BioRad, Hercules, Calif.) and values adjustedrelative to their respective loading control. Each sample was thennormalized relative to the value of the Notch3 ICD band from cellsplated on wells without ligand in the presence of hIgG control antibody.

The humanized 4F11 antibody variants significantly inhibitligand-induced cleavage of the Notch3 ICD as summarized in FIG. 11.

Example 12: Inhibition of Notch3-Dependent Transcription by Humanized4F11 Antibodies

Luciferase reporter assays were used to assess the ability of certainhumanized 4F11 antibodies to inhibit ligand-dependent Notch3 receptorsignaling and transcriptional activity. HCC1143 reporter linesco-cultured with CHO cells expressing Notch ligands were utilized forthese assays.

Stable lines expressing full length Notch ligands at the cell surfacewere produced by transfecting F1pIn™ CHO cells with Jag1, Jag2, DLL1, orDLL4 cDNAs cloned into the pcDNA5FRT vector using Lipofectamine 2000(Life Technologies) according to the manufacturer's protocol.Twenty-four hours after transfection, CHO cells were split into F12media containing 10% FBS, 2 mM L-Glutamine and 700 μg/ml hygromycin B(Sigma-Aldrich, St. Louis, Mo.) to select for transfected cells.Expression of Notch ligands was confirmed by FACS analysis usingantibodies against Jag1 (R&D Systems, Inc., Minneapolis, Minn.), Jag2(R&D Systems), DLL1 (R&D Systems), and DLL4 (BioLegend, San Diego,Calif.).

FlpIn™ CHO lines stably expressing Notch ligands were trypsinized,counted and seeded in 96-well plates at 60K cells/well in 100 μl ofHam's F12 K media without hygromycin B. The next day, HCC1143 reportercells were spun down and resuspended in RPMI media containing 2% FBS at0.2×10⁶ cells/ml. Antibodies were serially diluted into RPMI withoutFBS, and 100 μl of antibody solution was added to an equal volume ofreporter cells for 30 minutes at 37° C. in a 5% CO2 incubator. Afterremoving media from the hJag2 CHO cells, 100 μl of the HCC1143reporter/antibody mix was added to the ligand-expressing cells andallowed to incubate at 37° C. overnight. Twenty-four hours later,96-well plates placed at room temperature for 20 minutes, then processedwith the Bright Glo (Promega, Madison, Wis.) reporter assay protocol perthe manufacturer's instructions. Lysates were transferred towhite-walled 96 well plates (Greiner Bio-One, Frickenhausen, Germany)and read on a GloMax Luminometer (Promega) using the Bright Glo program.

As shown in FIG. 12A-B, the humanized 4F11 antibody variants inhibitactivation of ligand-induced transcription in the HCC1143 reporter cellline.

Example 13: Inhibition of Notch3 ICD Cleavage In Vivo

Selected humanized 4F11 antibodies (i.e., Hu4F11-70, Hu4F11-72,Hu4F11-78) were tested for their ability to inhibit cleavage of Notch3ICD expressed in HCC2429 tumors in vivo.

All mice were treated in accordance with the OLAW Public Health ServicePolicy on Human Care and Use of Laboratory Animals and the ILAR Guidefor the Care and Use of Laboratory Animals. All in vivo studies wereconducted following the protocols approved by the AVEO InstitutionalAnimal Care and Use Committee. HCC2429 is a lung cancer cell lineharboring a translocation of chromosome 19 that results inoverexpression of the Notch3 receptor. For the Notch3 ICD cleavageexperiments, approximately thirteen week old NCR nude mice (Taconic,Germantown, N.Y.) were inoculated subcutaneously into the right flankwith 5×10⁶ cells in 1:1 DMEM+Matrigel (Invitrogen, Carlsbad,Calif.)/Matrigel (BD Biosciences, San Jose, Calif.). Tumor measurementswere taken twice weekly, using vernier calipers. Tumor volume wascalculated using the formula: V=0.5×width×width×length. When tumorsapproached a volume of 300-400 mm³, mice were randomized into groups ofthree animals each (corresponds to mouse 1 (ml) through mouse 3 (m3), asdesignated in FIG. 13). The next day, mice were treated with 20 mg/kghIgG (control), or 20 mg/kg of antibodies Hu4F11-70, Hu4F11-72,Hu4F11-78 or murine 4F11 (mu4F11) by intraperitoneal injection. Micewere dosed once, and tumors were collected 24 hours later and snapfrozen.

To assess levels of cleaved Notch3 ICD, tumors were pulverized using aCovaris cryoPREP™ impactor (Covaris, Woburn, Mass.), resuspended in RIPAbuffer (Boston BioProducts) containing protease inhibitors, and rotatedat 4° C. for 1 hour. Lysates were clarified by spinning at 14k rpm for15 minutes in a refrigerated microcentrifuge. Protein concentration foreach sample was measured using the BioRad DC protein assay (BioRad).Equal concentrations of protein from each sample were loaded onto an SDSPAGE gel and transferred onto nitrocellulose by Western blotting. Blotswere probed with antibody against the C-terminus of Notch3 (CellSignaling) to detect levels of the cleaved ICD. Blots were also probedwith antibody against β-tubulin (Cell Signaling) for use as a loadingcontrol. Bands were quantified using Image Lab 3.0 software (BioRad),and the intensity of Notch3 ICD bands were normalized to theirrespective β-tubulin loading control. All humanized 4F11 antibodiestested (i.e., Hu4F11-70, Hu4F11-72, Hu4F11-78) significantly inhibitNotch3 activation in vivo, as measured by Notch3 ICD levels present intumors 24 hours after single dose antibody treatment (FIG. 13).

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andthe range of equivalency of the claims are intended to be embracedtherein.

What is claimed is:
 1. An isolated nucleic acid comprising a nucleotidesequence encoding an immunoglobulin heavy chain variable region selectedfrom the group consisting of: (a) an immunoglobulin heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:36 (Sh4F11 Hv3-23A28T S31H T62S); (b) an immunoglobulin heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:2 (4F11, Ch4F11Chimeric); (c) an immunoglobulin heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO:34 (Sh4F11 Hv3-23); (d) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:38 (Sh4F11 Hv3-23 S31H T62S); (e) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:40 (Sh4F11 Hv3-23 A28T S31N T62S); and (f) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:42 (Sh4F11 Hv3-23 A28T T62S).
 2. An expressionvector comprising the nucleic acid of claim
 1. 3. A host cell comprisingthe expression vector of claim
 2. 4. A method of producing a polypeptidecomprising an immunoglobulin heavy chain variable region, the methodcomprising: (a) growing the host cell of claim 3 under conditions sothat the host cell expresses the polypeptide comprising theimmunoglobulin heavy chain variable region; and (b) purifying thepolypeptide comprising the immunoglobulin heavy chain variable region.5. A method of inhibiting or reducing proliferation of a tumor cellcomprising exposing the cell to an effective amount of an antibody thatbinds human Notch3 to inhibit or reduce proliferation of the tumor cell,wherein the antibody comprises: an immunoglobulin heavy chain variableregion and an immunoglobulin light chain variable region selected fromthe group consisting of: (a) an immunoglobulin heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:36 (Sh4F11 Hv3-23A28T S31H T62S), and an immunoglobulin light chain variable regioncomprising the amino acid sequence of SEQ ID NO:46 (Hu4F11 Kv2D-29N28H); (b) an immunoglobulin heavy chain variable region comprising theamino acid sequence of SEQ ID NO:2 (4F11, Ch4F11 Chimeric), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:4 (4F11, Ch4F11 Chimeric); (c) an immunoglobulinheavy chain variable region comprising the amino acid sequence of SEQ IDNO:2 (4F11, Ch4F11 Chimeric), and an immunoglobulin light chain variableregion comprising the amino acid sequence of SEQ ID NO:44 (Hu4F11Kv2D-29); (d) an immunoglobulin heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO:34 (Sh4F11 Hv3-23), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:4 (4F11, Ch4F11 Chimeric); (e) an immunoglobulinheavy chain variable region comprising the amino acid sequence of SEQ IDNO:34 (Sh4F11 Hv3-23), and an immunoglobulin light chain variable regioncomprising the amino acid sequence of SEQ ID NO:44 (Hu4F11 Kv2D-29); (f)an immunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:38 (Sh4F11 Hv3-23 S31H T62S), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:46 (Hu4F11 Kv2D-29 N28H), (g) an immunoglobulinheavy chain variable region comprising the amino acid sequence of SEQ IDNO:40 (Sh4F11 Hv3-23 A28T S31N T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:46(Hu4F11 Kv2D-29 N28H), (h) an immunoglobulin heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:42 (Sh4F11 Hv3-23 A28TT62S), and an immunoglobulin light chain variable region comprising theamino acid sequence of SEQ ID NO:46 (Hu4F11 Kv2D-29 N28H), (i) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:36 (Sh4F11 Hv3-23 A28T S31H T62S), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:48 (Hu4F11 Kv2D-29 N28Q), (j) an immunoglobulinheavy chain variable region comprising the amino acid sequence of SEQ IDNO:38 (Sh4F11 Hv3-23 S31H T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:48(Hu4F11 Kv2D-29 N28Q), (k) an immunoglobulin heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:40 (Sh4F11 Hv3-23 A28TS31N T62S), and an immunoglobulin light chain variable region comprisingthe amino acid sequence of SEQ ID NO:48 (Hu4F11 Kv2D-29 N28Q), (l) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:42 (Sh4F11 Hv3-23 A28T T62S), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:48 (Hu4F11 Kv2D-29 N28Q), (m) an immunoglobulinheavy chain variable region comprising the amino acid sequence of SEQ IDNO:36 (Sh4F11 Hv3-23 A28T S31H T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:50(Hu4F11 Kv2D-29 N28Y); (n) an immunoglobulin heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:38 (Sh4F11 Hv3-23 S31HT62S), and an immunoglobulin light chain variable region comprising theamino acid sequence of SEQ ID NO:50 (Hu4F11 Kv2D-29 N28Y); (o) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:40 (Sh4F11 Hv3-23 A28T S31N T62S), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:50 (Hu4F11 Kv2D-29 N28Y); and (p) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:42 (Sh4F11 Hv3-23 A28T T62S), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:50 (Hu4F11 Kv2D-29 N28Y); or an immunoglobulinheavy chain and an immunoglobulin light chain selected from the groupconsisting of: (q) an immunoglobulin heavy chain comprising the aminoacid sequence of SEQ ID NO: 78 (Sh4F11 Hv3-23 A28T S31H T62S), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 90 (Hu4F11 Kv2D-29 N28H Kappa); (r) an immunoglobulin heavy chaincomprising the amino acid sequence of SEQ ID NO: 27 (4F11), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 29 (4F11); (s) an immunoglobulin heavy chain comprising the aminoacid sequence of SEQ ID NO: 74 (Ch4F11), and an immunoglobulin lightchain comprising the amino acid sequence of SEQ ID NO: 86 (Ch4F11); (t)an immunoglobulin heavy chain comprising the amino acid sequence of SEQID NO: 74 (Ch4F11), and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO: 88 (Hu4F11 Kv2D-29); (u) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 76 (Sh4F11 Hv3-23), and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO: 86 (Ch4F11); (v) an immunoglobulinheavy chain comprising the amino acid sequence of SEQ ID NO: 76 (Sh4F11Hv3-23), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 88 (Hu4F11 Kv2D-29); (w) an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 80 (Sh4F11 Hv3-23S31H T62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 90 (Hu4F11 Kv2D-29 N28H Kappa); (x) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 82 (Sh4F11 Hv3-23 A28T S31N T62S), and an immunoglobulin light chaincomprising the amino acid sequence of SEQ ID NO: 90 (Hu4F11 Kv2D-29 N28HKappa); (y) an immunoglobulin heavy chain comprising the amino acidsequence of SEQ ID NO: 84 (Sh4F11 Hv3-23 A28T T62S), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 90 (Hu4F11 Kv2D-29 N28H Kappa); (z) an immunoglobulin heavy chaincomprising the amino acid sequence of SEQ ID NO: 78 (Sh4F11 Hv3-23 A28TS31H T62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 92 (Hu4F11 Kv2D-29 N28Q Kappa); (aa) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 80 (Sh4F11 Hv3-23 S31H T62S), and an immunoglobulin light chaincomprising the amino acid sequence of SEQ ID NO: 92 (Hu4F11 Kv2D-29 N28QKappa); (bb) an immunoglobulin heavy chain comprising the amino acidsequence of SEQ ID NO: 82 (Sh4F11 Hv3-23 A28T S31N T62S), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 92 (Hu4F11 Kv2D-29 N28Q Kappa); (cc) an immunoglobulin heavy chaincomprising the amino acid sequence of SEQ ID NO: 84 (Sh4F11 Hv3-23 A28TT62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 92 (Hu4F11 Kv2D-29 N28Q Kappa); (dd) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 78 (Sh4F11 Hv3-23 A28T S31H T62S), and an immunoglobulin light chaincomprising the amino acid sequence of SEQ ID NO: 94 (Hu4F11 Kv2D-29 N28YKappa); (ee) an immunoglobulin heavy chain comprising the amino acidsequence of SEQ ID NO: 80 (Sh4F11 Hv3-23 S31H T62S), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 94 (Hu4F11 Kv2D-29 N28Y Kappa); (ff) an immunoglobulin heavy chaincomprising the amino acid sequence of SEQ ID NO: 82 (Sh4F11 Hv3-23 A28TS31N T62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 94 (Hu4F11 Kv2D-29 N28Y Kappa); and (gg) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 84 (Sh4F11 Hv3-23 A28T T62S), and an immunoglobulin light chaincomprising the amino acid sequence of SEQ ID NO: 94 (Hu4F11 Kv2D-29 N28YKappa).
 6. A method of inhibiting or reducing tumor growth in a mammal,the method comprising exposing the mammal to an effective amount of anantibody that binds human Notch3 to inhibit or reduce growth of thetumor, wherein the antibody comprises: an immunoglobulin heavy chainvariable region and an immunoglobulin light chain variable regionselected from the group consisting of: (a) an immunoglobulin heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:36(Sh4F11 Hv3-23 A28T S31H T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:46(Hu4F11 Kv2D-29 N28H); (b) an immunoglobulin heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:2 (4F11, Ch4F11Chimeric), and an immunoglobulin light chain variable region comprisingthe amino acid sequence of SEQ ID NO:4 (4F11, Ch4F11 Chimeric); (c) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:2 (4F11, Ch4F11 Chimeric), and an immunoglobulinlight chain variable region comprising the amino acid sequence of SEQ IDNO:44 (Hu4F11 Kv2D-29); (d) an immunoglobulin heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:34 (Sh4F11Hv3-23), and an immunoglobulin light chain variable region comprisingthe amino acid sequence of SEQ ID NO:4 (4F11, Ch4F11 Chimeric); (e) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:34 (Sh4F11 Hv3-23), and an immunoglobulin lightchain variable region comprising the amino acid sequence of SEQ ID NO:44(Hu4F11 Kv2D-29); (f) an immunoglobulin heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:38 (Sh4F11 Hv3-23 S31HT62S), and an immunoglobulin light chain variable region comprising theamino acid sequence of SEQ ID NO:46 (Hu4F11 Kv2D-29 N28H); (g) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:40 (Sh4F11 Hv3-23 A28T S31N T62S), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:46 (Hu4F11 Kv2D-29 N28H); (h) an immunoglobulinheavy chain variable region comprising the amino acid sequence of SEQ IDNO:42 (Sh4F11 Hv3-23 A28T T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:46(Hu4F11 Kv2D-29 N28H); (i) an immunoglobulin heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:36 (Sh4F11 Hv3-23 A28TS31H T62S), and an immunoglobulin light chain variable region comprisingthe amino acid sequence of SEQ ID NO:48 (Hu4F11 Kv2D-29 N28Q); (j) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:38 (Sh4F11 Hv3-23 S31H T62S), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:48 (Hu4F11 Kv2D-29 N28Q); (k) an immunoglobulinheavy chain variable region comprising the amino acid sequence of SEQ IDNO:40 (Sh4F11 Hv3-23 A28T S31N T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:48(Hu4F11 Kv2D-29 N28Q), (l) an immunoglobulin heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:42 (Sh4F11 Hv3-23 A28TT62S), and an immunoglobulin light chain variable region comprising theamino acid sequence of SEQ ID NO:48 (Hu4F11 Kv2D-29 N28Q), (m) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:36 (Sh4F11 Hv3-23 A28T S31H T62S), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:50 (Hu4F11 Kv2D-29 N28Y), (n) an immunoglobulinheavy chain variable region comprising the amino acid sequence of SEQ IDNO:38 (Sh4F11 Hv3-23 S31H T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:50(Hu4F11 Kv2D-29 N28Y), (o) an immunoglobulin heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:40 (Sh4F11 Hv3-23 A28TS31N T62S), and an immunoglobulin light chain variable region comprisingthe amino acid sequence of SEQ ID NO:50 (Hu4F11 Kv2D-29 N28Y), and (p)an immunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:42 (Sh4F11 Hv3-23 A28T T62S), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:50 (Hu4F11 Kv2D-29 N28Y), or an immunoglobulinheavy chain and an immunoglobulin light chain selected from the groupconsisting of: (q) an immunoglobulin heavy chain comprising the aminoacid sequence of SEQ ID NO: 78 (Sh4F11 Hv3-23 A28T S31H T62S), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 90 (Hu4F11 Kv2D-29 N28H Kappa); (r) an immunoglobulin heavy chaincomprising the amino acid sequence of SEQ ID NO: 27 (4F11), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 29 (4F11); (s) an immunoglobulin heavy chain comprising the aminoacid sequence of SEQ ID NO: 74 (Ch4F11), and an immunoglobulin lightchain comprising the amino acid sequence of SEQ ID NO: 86 (Ch4F11); (t)an immunoglobulin heavy chain comprising the amino acid sequence of SEQID NO: 74 (Ch4F11), and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO: 88 (Hu4F11 Kv2D-29); (u) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 76 (Sh4F11 Hv3-23), and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO: 86 (Ch4F11); (v) an immunoglobulinheavy chain comprising the amino acid sequence of SEQ ID NO: 76 (Sh4F11Hv3-23), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 88 (Hu4F11 Kv2D-29); (w) an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 80 (Sh4F11 Hv3-23S31H T62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 90 (Hu4F11 Kv2D-29 N28H Kappa); (x) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 82 (Sh4F11 Hv3-23 A28T S31N T62S), and an immunoglobulin light chaincomprising the amino acid sequence of SEQ ID NO: 90 (Hu4F11 Kv2D-29 N28HKappa); (y) an immunoglobulin heavy chain comprising the amino acidsequence of SEQ ID NO: 84 (Sh4F11 Hv3-23 A28T T62S), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 90 (Hu4F11 Kv2D-29 N28H Kappa); (z) an immunoglobulin heavy chaincomprising the amino acid sequence of SEQ ID NO: 78 (Sh4F11 Hv3-23 A28TS31H T62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 92 (Hu4F11 Kv2D-29 N28Q Kappa); (aa) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 80 (Sh4F11 Hv3-23 S31H T62S), and an immunoglobulin light chaincomprising the amino acid sequence of SEQ ID NO: 92 (Hu4F11 Kv2D-29 N28QKappa); (bb) an immunoglobulin heavy chain comprising the amino acidsequence of SEQ ID NO: 82 (Sh4F11 Hv3-23 A28T S31N T62S), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 92 (Hu4F11 Kv2D-29 N28Q Kappa); (cc) an immunoglobulin heavy chaincomprising the amino acid sequence of SEQ ID NO: 84 (Sh4F11 Hv3-23 A28TT62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 92 (Hu4F11 Kv2D-29 N28Q Kappa); (dd) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 78 (Sh4F11 Hv3-23 A28T S31H T62S), and an immunoglobulin light chaincomprising the amino acid sequence of SEQ ID NO: 94 (Hu4F11 Kv2D-29 N28YKappa); (ee) an immunoglobulin heavy chain comprising the amino acidsequence of SEQ ID NO: 80 (Sh4F11 Hv3-23 S31H T62S), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 94 (Hu4F11 Kv2D-29 N28Y Kappa); (ff) an immunoglobulin heavy chaincomprising the amino acid sequence of SEQ ID NO: 82 (Sh4F11 Hv3-23 A28TS31N T62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 94 (Hu4F11 Kv2D-29 N28Y Kappa); and (gg) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 84 (Sh4F11 Hv3-23 A28T T62S), and an immunoglobulin light chaincomprising the amino acid sequence of SEQ ID NO: 94 (Hu4F11 Kv2D-29 N28YKappa).
 7. A method of treating cancer in a human subject, the methodcomprising administering to the subject an effective amount of anantibody that binds human Notch3 to a human subject in need thereof,wherein the antibody comprises: an immunoglobulin heavy chain variableregion and an immunoglobulin light chain variable region selected fromthe group consisting of: (a) an immunoglobulin heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:36 (Sh4F11 Hv3-23A28T S31H T62S), and an immunoglobulin light chain variable regioncomprising the amino acid sequence of SEQ ID NO:46 (Hu4F11 Kv2D-29N28H); (b) an immunoglobulin heavy chain variable region comprising theamino acid sequence of SEQ ID NO:2 (4F11, Ch4F11 Chimeric), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:4 (4F11, Ch4F11 Chimeric); (c) an immunoglobulinheavy chain variable region comprising the amino acid sequence of SEQ IDNO:2 (4F11, Ch4F11 Chimeric), and an immunoglobulin light chain variableregion comprising the amino acid sequence of SEQ ID NO:44 (Hu4F11Kv2D-29); (d) an immunoglobulin heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO:34 (Sh4F11 Hv3-23), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:4 (4F11, Ch4F11 Chimeric); (e) an immunoglobulinheavy chain variable region comprising the amino acid sequence of SEQ IDNO:34 (Sh4F11 Hv3-23), and an immunoglobulin light chain variable regioncomprising the amino acid sequence of SEQ ID NO:44 (Hu4F11 Kv2D-29); (f)an immunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:38 (Sh4F11 Hv3-23 S31H T62S), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:46 (Hu4F11 Kv2D-29 N28H); (g) an immunoglobulinheavy chain variable region comprising the amino acid sequence of SEQ IDNO:40 (Sh4F11 Hv3-23 A28T S31N T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:46(Hu4F11 Kv2D-29 N28H); (h) an immunoglobulin heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:42 (Sh4F11 Hv3-23 A28TT62S), and an immunoglobulin light chain variable region comprising theamino acid sequence of SEQ ID NO:46 (Hu4F11 Kv2D-29 N28H); (i) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:36 (Sh4F11 Hv3-23 A28T S31H T62S), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:48 (Hu4F11 Kv2D-29 N28Q); (j) an immunoglobulinheavy chain variable region comprising the amino acid sequence of SEQ IDNO:38 (Sh4F11 Hv3-23 S31H T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:48(Hu4F11 Kv2D-29 N28Q); (k) an immunoglobulin heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:40 (Sh4F11 Hv3-23 A28TS31N T62S), and an immunoglobulin light chain variable region comprisingthe amino acid sequence of SEQ ID NO:48 (Hu4F11 Kv2D-29 N28Q); (l) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:42 (Sh4F11 Hv3-23 A28T T62S), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:48 (Hu4F11 Kv2D-29 N28Q); (m) an immunoglobulinheavy chain variable region comprising the amino acid sequence of SEQ IDNO:36 (Sh4F11 Hv3-23 A28T S31H T62S), and an immunoglobulin light chainvariable region comprising the amino acid sequence of SEQ ID NO:50(Hu4F11 Kv2D-29 N28Y); (n) an immunoglobulin heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:38 (Sh4F11 Hv3-23 S31HT62S), and an immunoglobulin light chain variable region comprising theamino acid sequence of SEQ ID NO:50 (Hu4F11 Kv2D-29 N28Y); (o) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:40 (Sh4F11 Hv3-23 A28T S31N T62S), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:50 (Hu4F11 Kv2D-29 N28Y); and (p) animmunoglobulin heavy chain variable region comprising the amino acidsequence of SEQ ID NO:42 (Sh4F11 Hv3-23 A28T T62S), and animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:50 (Hu4F11 Kv2D-29 N28Y); or an immunoglobulinheavy chain and an immunoglobulin light chain selected from the groupconsisting of: (q) an immunoglobulin heavy chain comprising the aminoacid sequence of SEQ ID NO: 78 (Sh4F11 Hv3-23 A28T S31H T62S), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 90 (Hu4F11 Kv2D-29 N28H Kappa); (r) an immunoglobulin heavy chaincomprising the amino acid sequence of SEQ ID NO: 27 (4F11), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 29 (4F11); (s) an immunoglobulin heavy chain comprising the aminoacid sequence of SEQ ID NO: 74 (Ch4F11), and an immunoglobulin lightchain comprising the amino acid sequence of SEQ ID NO: 86 (Ch4F11); (t)an immunoglobulin heavy chain comprising the amino acid sequence of SEQID NO: 74 (Ch4F11), and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO: 88 (Hu4F11 Kv2D-29); (u) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 76 (Sh4F11 Hv3-23), and an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO: 86 (Ch4F11); (v) an immunoglobulinheavy chain comprising the amino acid sequence of SEQ ID NO: 76 (Sh4F11Hv3-23), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 88 (Hu4F11 Kv2D-29); (w) an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 80 (Sh4F11 Hv3-23S31H T62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 90 (Hu4F11 Kv2D-29 N28H Kappa); (x) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 82 (Sh4F11 Hv3-23 A28T S31N T62S), and an immunoglobulin light chaincomprising the amino acid sequence of SEQ ID NO: 90 (Hu4F11 Kv2D-29 N28HKappa); (y) an immunoglobulin heavy chain comprising the amino acidsequence of SEQ ID NO: 84 (Sh4F11 Hv3-23 A28T T62S), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 90 (Hu4F11 Kv2D-29 N28H Kappa); (z) an immunoglobulin heavy chaincomprising the amino acid sequence of SEQ ID NO: 78 (Sh4F11 Hv3-23 A28TS31H T62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 92 (Hu4F11 Kv2D-29 N28Q Kappa); (aa) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 80 (Sh4F11 Hv3-23 S31H T62S), and an immunoglobulin light chaincomprising the amino acid sequence of SEQ ID NO: 92 (Hu4F11 Kv2D-29 N28QKappa); (bb) an immunoglobulin heavy chain comprising the amino acidsequence of SEQ ID NO: 82 (Sh4F11 Hv3-23 A28T S31N T62S), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 92 (Hu4F11 Kv2D-29 N28Q Kappa); (cc) an immunoglobulin heavy chaincomprising the amino acid sequence of SEQ ID NO: 84 (Sh4F11 Hv3-23 A28TT62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 92 (Hu4F11 Kv2D-29 N28Q Kappa); (dd) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 78 (Sh4F11 Hv3-23 A28T S31H T62S), and an immunoglobulin light chaincomprising the amino acid sequence of SEQ ID NO: 94 (Hu4F11 Kv2D-29 N28YKappa); (ee) an immunoglobulin heavy chain comprising the amino acidsequence of SEQ ID NO: 80 (Sh4F11 Hv3-23 S31H T62S), and animmunoglobulin light chain comprising the amino acid sequence of SEQ IDNO: 94 (Hu4F11 Kv2D-29 N28Y Kappa); (ff) an immunoglobulin heavy chaincomprising the amino acid sequence of SEQ ID NO: 82 (Sh4F11 Hv3-23 A28TS31N T62S), and an immunoglobulin light chain comprising the amino acidsequence of SEQ ID NO: 94 (Hu4F11 Kv2D-29 N28Y Kappa); and (gg) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 84 (Sh4F11 Hv3-23 A28T T62S), and an immunoglobulin light chaincomprising the amino acid sequence of SEQ ID NO: 94 (Hu4F11 Kv2D-29 N28YKappa).
 8. An isolated antibody that competes with an antibodycomprising an immunoglobulin heavy chain variable region comprising theamino acid sequence of SEQ ID NO:36 (Sh4F11 Hv3-23 A28T S31H T62S) andan immunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:46 (Hu4F11 Kv2D-29 N28H) for binding to EGF-likerepeats 1-11 of human Notch3, wherein the isolated antibody comprises animmunoglobulin heavy chain variable region comprising an amino acidsequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO:36 (Sh4F11 Hv3-23 A28T S31H T62S) and an immunoglobulin lightchain variable region comprising an amino acid sequence that is at least95% identical to the amino acid sequence of SEQ ID NO:46 (Hu4F11 Kv2D-29N28H).
 9. The isolated antibody of claim 8, wherein the isolatedantibody comprises an immunoglobulin heavy chain variable regioncomprising an amino acid sequence that is at least 98% identical to theamino acid sequence of SEQ ID NO:36 (Sh4F11 Hv3-23 A28T S31H T62S) andan immunoglobulin light chain variable region comprising an amino acidsequence that is at least 98% identical to the amino acid sequence ofSEQ ID NO:46 (Hu4F11 Kv2D-29 N28H)).
 10. The isolated antibody of claim8, wherein the isolated antibody comprises an immunoglobulin heavy chainvariable region comprising an amino acid sequence that is at least 99%identical to the amino acid sequence of SEQ ID NO:36 (Sh4F11 Hv3-23 A28TS31H T62S) and an immunoglobulin light chain variable region comprisingan amino acid sequence that is at least 99% identical to the amino acidsequence of SEQ ID NO:46 (Hu4F11 Kv2D-29 N281).
 11. An isolated nucleicacid comprising a nucleotide sequence encoding an immunoglobulin lightchain variable region selected from the group consisting of: (a) animmunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:46 (Hu4F11 Kv2D-29 N28H); (b) an immunoglobulinlight chain variable region comprising the amino acid sequence of SEQ IDNO:4 (4F11, Ch4F11 Chimeric); (c) an immunoglobulin light chain variableregion comprising the amino acid sequence of SEQ ID NO:44 (Hu4F11Kv2D-29); (d) an immunoglobulin light chain variable region comprisingthe amino acid sequence of SEQ ID NO:48 (Hu4F11 Kv2D-29 N28Q); and (e)an immunoglobulin light chain variable region comprising the amino acidsequence of SEQ ID NO:50 (Hu4F11 Kv2D-29 N28Y).
 12. An expression vectorcomprising the nucleic acid of claim
 11. 13. A host cell comprising theexpression vector of claim
 12. 14. A method of producing a polypeptidecomprising an immunoglobulin light chain variable region, the methodcomprising: (a) growing the host cell of claim 13 under conditions sothat the host cell expresses the polypeptide comprising theimmunoglobulin light chain variable region; and (b) purifying thepolypeptide comprising the immunoglobulin light chain variable region.15. The expression vector of claim 2, further comprising the nucleicacid of claim
 11. 16. A host cell comprising the expression vector ofclaim
 15. 17. A method of producing an antibody that binds human Notch3or an antigen-binding fragment of the antibody, the method comprising:(a) growing the host cell of claim 16 under conditions so that the hostcell expresses a polypeptide or polypeptides comprising theimmunoglobulin heavy chain variable region and the immunoglobulin lightchain variable region, thereby producing the antibody or the antigenbinding-fragment of the antibody; and (b) purifying the antibody or theantigen-binding fragment of the antibody.
 18. An isolated nucleic acidcomprising a nucleotide sequence encoding an immunoglobulin heavy chainselected from the group consisting of: (a) an immunoglobulin heavy chaincomprising the amino acid sequence of SEQ ID NO: 78 (Sh4F11 Hv3-23 A28TS31H T62S); (b) an immunoglobulin heavy chain comprising the amino acidsequence of SEQ ID NO: 27 (4F11); (c) an immunoglobulin heavy chaincomprising the amino acid sequence of SEQ ID NO: 74 (Ch4F11); (d) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 76 (Sh4F11 Hv3-23); (e) an immunoglobulin heavy chain comprising theamino acid sequence of SEQ ID NO: 80 (Sh4F11 Hv3-23 S31H T62S); (f) animmunoglobulin heavy chain comprising the amino acid sequence of SEQ IDNO: 82 (Sh4F11 Hv3-23 A28T S31N T62S); and (g) an immunoglobulin heavychain comprising the amino acid sequence of SEQ ID NO: 84 (Sh4F11 Hv3-23A28T T62S).
 19. An expression vector comprising the nucleic acid ofclaim
 18. 20. A host cell comprising the expression vector of claim 19.21. A method of producing a polypeptide comprising an immunoglobulinheavy chain, the method comprising: (a) growing the host cell of claim20 under conditions so that the host cell expresses the polypeptidecomprising the immunoglobulin heavy chain; and (b) purifying thepolypeptide comprising the immunoglobulin heavy chain.
 22. An isolatednucleic acid comprising a nucleotide sequence encoding an immunoglobulinlight chain selected from the group consisting of: (a) an immunoglobulinlight chain comprising the amino acid sequence of SEQ ID NO: 90 (Hu4F11Kv2D-29 N28H Kappa); (b) an immunoglobulin light chain comprising theamino acid sequence of SEQ ID NO: 29 (4F11); (c) an immunoglobulin lightchain comprising the amino acid sequence of SEQ ID NO: 86 (Ch4F11); (d)an immunoglobulin light chain comprising the amino acid sequence of SEQID NO: 88 (Hu4F11 Kv2D-29); (e) an immunoglobulin light chain comprisingthe amino acid sequence of SEQ ID NO: 92 (Hu4F11 Kv2D-29 N28Q Kappa);and (f) an immunoglobulin light chain comprising the amino acid sequenceof SEQ ID NO: 94 (Hu4F11 Kv2D-29 N28Y Kappa).
 23. An expression vectorcomprising the nucleic acid of claim
 22. 24. A host cell comprising theexpression vector of claim
 23. 25. A method of producing a polypeptidecomprising an immunoglobulin light chain, the method comprising: (a)growing the host cell of claim 24 under conditions so that the host cellexpresses the polypeptide comprising the immunoglobulin light chain; and(b) purifying the polypeptide comprising the immunoglobulin light chain.26. The expression vector of claim 19, further comprising the nucleicacid of claim
 22. 27. A host cell comprising the expression vector ofclaim
 26. 28. A method of producing an antibody that binds human Notch3or an antigen-binding fragment of the antibody, the method comprising:(a) growing the host cell of claim 27 under conditions so that the hostcell expresses a polypeptide or polypeptides comprising theimmunoglobulin heavy chain and the immunoglobulin light chain, therebyproducing the antibody or the antigen-binding fragment of the antibody;and (b) purifying the antibody or the antigen-binding fragment of theantibody.